U.S. patent application number 11/158505 was filed with the patent office on 2006-01-05 for optimized dosing with anti-cd4 antibodies for tolerance induction in primates.
This patent application is currently assigned to TolerRX, Inc.. Invention is credited to Paul Ponath, Patricia Rao, Douglas J.V Ringler, Dawn Winsor-Hines.
Application Number | 20060002921 11/158505 |
Document ID | / |
Family ID | 35407000 |
Filed Date | 2006-01-05 |
United States Patent
Application |
20060002921 |
Kind Code |
A1 |
Winsor-Hines; Dawn ; et
al. |
January 5, 2006 |
Optimized dosing with anti-CD4 antibodies for tolerance induction
in primates
Abstract
The present invention is based, at least in part, on the finding
that tolerance can be induced by inhibition of CD4+ cells (and
optionally CD8+ cells). Accordingly, the optimized dosing methods
of the invention are useful in treating a primate, e.g., a human,
by inhibiting CD4+ T cells to induce tolerance to at least one
antigen, e.g., self or foreign, such as for inducting tolerance in
a primate against a soluble or a cell bound antigen (e.g., an
allogeneic or xenogeneic transplanted antigen).
Inventors: |
Winsor-Hines; Dawn;
(Framingham, MA) ; Rao; Patricia; (Acton, MA)
; Ringler; Douglas J.V; (Boston, MA) ; Ponath;
Paul; (San Francisco, CA) |
Correspondence
Address: |
LAHIVE & COCKFIELD, LLP.
28 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
TolerRX, Inc.
Cambridge
MA
|
Family ID: |
35407000 |
Appl. No.: |
11/158505 |
Filed: |
June 21, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60582181 |
Jun 22, 2004 |
|
|
|
Current U.S.
Class: |
424/141.1 ;
424/144.1 |
Current CPC
Class: |
A61K 2039/505 20130101;
C07K 16/2812 20130101; C07K 2317/56 20130101; C07K 16/2815
20130101; C07K 2317/41 20130101; C07K 2317/75 20130101; A61P 37/06
20180101; A61P 43/00 20180101; A61P 37/00 20180101; C07K 2317/53
20130101; C07K 2317/24 20130101; C07K 2317/52 20130101 |
Class at
Publication: |
424/141.1 ;
424/144.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395 |
Claims
1. A method for treating a primate to induce tolerance to at least
one foreign antigen comprising, administering to the primate at
least one anti-CD4 antibody or CD4 binding fragment thereof,
wherein the at least one anti-CD4 antibody is administered at a
dose of between about 20 mg/kg to 40 mg/kg in at least three
separate doses.
2. The method of claim 1, wherein the at least one anti-CD4
antibody is administered at a dose of about 20 mg/kg.
3. The method of claim 1, wherein the at least one anti-CD4
antibody is administered in at least four separate doses.
4. The method of claim 1, wherein the at least one anti-CD4
antibody is administered in at least five separate doses.
5. The method of claim 1, wherein the at least one anti-CD4
antibody is administered on at least days -1, 3 or 4, 8 and 12
relative to administration of the foreign antigen.
6. The method of claim 1, wherein the at least one anti-CD4
antibody is administered on at least days -1, 1, and 3 relative to
administration of the foreign antigen.
7. The method of claim 1, wherein the foreign antigen is a soluble
antigen.
8. The method of claim 1, wherein the first dose of the at least
one anti-CD4 antibody is administered prior to administration of
the foreign antigen.
9. The method of claim 1, wherein the at least one anti-CD4
antibody is humanized and modified to reduce Fc receptor and
complement binding.
10. A method for treating a primate to induce tolerance to at least
one foreign antigen comprising, administering to the primate at
least one anti-CD4 antibody or CD4 binding fragment thereof,
wherein at least one dose of the at least one anti-CD4 antibody is
administered at least one day prior to administration of the
foreign antigen.
11. A method for treating a primate to induce tolerance to at least
one foreign antigen comprising, administering to the primate at
least one anti-CD4 antibody or CD4 binding fragment thereof,
wherein the at least one anti-CD4 antibody is administered at a
dose sufficient to maintain a serum concentration of anti-CD4
antibody at a level of about 20 .mu.g/ml during the tolerance
induction phase.
12. The method of claim 11, wherein at least one dose of the at
least one anti-CD4 antibody is administered one day prior to
administration of the foreign antigen.
13. The method of claim 11, wherein the at least one anti-CD4
antibody is administered at a dose of between about 20 mg/kg and 40
mg/kg.
14. A method for treating a primate to induce tolerance to at least
one foreign antigen comprising, administering to the primate at
least one anti-CD4 antibody or CD4 binding fragment thereof,
wherein the at least one anti-CD4 antibody is administered at a
dose sufficient to achieve about 85% saturation of CD4 sites on T
cells in the primate during the tolerance induction phase.
15. The method of claim 14, wherein the at least one anti-CD4
antibody is not administered for more than about two weeks.
16. The method of claim 14, wherein at least one dose of the at
least one anti-CD4 antibody is administered one day prior to
administration of the foreign antigen.
17. The method of claim 14, wherein the at least one anti-CD4
antibody is administered at a dose of between about 20 mg/kg and 40
mg/kg.
18. The method of claim 14, wherein the at least one anti-CD4
antibody is humanized and modified to reduce Fc receptor and
complement binding.
19. A method for inducing tolerance in a primate to a soluble
antigen, comprising, administering to a primate at least one
anti-CD4 antibody or CD4 binding fragment thereof, wherein the at
least one anti-CD4 antibody is administered at a dose of between
about 20-40 mg/kg in at least three separate doses, such that
tolerance to the soluble antigen is induced.
20. The method of claim 19, wherein at least one dose of the at
least one anti-CD4 antibody is administered one day prior to
administration of the soluble antigen.
21. The method of claim 19, wherein the at least one anti-CD4
antibody is administered at a dose of about 20 mg/kg.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application 60/582,181, filed Jun. 22, 2004, titled "Optimized
Dosing of Anti-CD4 Antibodies for Tolerance Inducing Induction in
Primates". This application is related to U.S. Provisional
Application 60/431,839, filed Dec. 9, 2002, titled "Introducing
Tolerance to Proteins in Primates," and U.S. Ser. No. 10/731,984
"Introducing Tolerance in Primates filed Dec. 9, 2003. The entire
contents of each of these applications are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] This invention relates to tolerance induction and more
particularly to inducing tolerance in a primate against an
antigen(s) and in particular a foreign antigen.
[0003] There have been numerous attempts to induce tolerance
against foreign and self antigens in primates. For example, in the
field of transplantation, there is a need to induce tolerance to
foreign antigens in a transplant so as to prevent its rejection. At
present, rejection can only be prevented by the use of long-term
(chronic) immunosuppression which carries risks of infection,
cancer and drug toxicity.
[0004] In addition, in the treatment of a patient with a
therapeutic protein, in many cases, treatment becomes less
effective or totally ineffective as a result of an immune response
to that foreign protein.
[0005] Although success has been demonstrated using anti-CD4
antibodies in rodents, tolerance induction with anti-CD4 antibodies
has yet to be demonstrated in primates. As a result, there is a
need for a treatment that induces tolerance to antigen(s), e.g.,
cell bound or soluble proteins that reduces or eliminates the need
for immunosuppressive drugs and long term immune suppression in a
primate against an antigen(s) and in particular a foreign
protein(s).
SUMMARY OF THE INVENTION
[0006] The present invention advances the art by providing
optimized doses of anti-CD4 that are able to reduce immune
responses to foreign proteins without long term immunosuppression.
In accordance with an aspect of the present invention, there is
provided a process for tolerizing a primate against an antigen(s)
by use of a CD4 antibody or fragment thereof in an amount and for a
time effective to induce tolerance against at least one antigen. In
one embodiment, the anti-CD4 antibody is administered in
combination with a second agent that promotes tolerance. In one
embodiment, the second agent is an anti-CD8 antibody or other agent
that inhibits the activity of CD8+ cells.
[0007] In one aspect, the invention is directed to a method for
treating a primate to induce tolerance to at least one foreign
antigen comprising, administering to the primate at least one
anti-CD4 antibody or CD4 binding fragment thereof, wherein the at
least one anti-CD4 antibody is administered at a dose of between
about 20 mg/kg to 40 mg/kg in at least three separate doses.
[0008] In one embodiment, the at least one anti-CD4 antibody is
administered at a dose of about 20 mg/kg.
[0009] In another embodiment, the at least one anti-CD4 antibody is
administered in at least four separate doses. In another
embodiment, the at least one anti-CD4 antibody is administered in
at least five separate doses.
[0010] In one embodiment, the at least one anti-CD4 antibody is
administered on at least days -1, 3 or 4, 8 and 12 relative to
administration of the foreign antigen.
[0011] In one embodiment, wherein the at least one anti-CD4
antibody is administered on at least days -1, 1, and 3 relative to
administration of the foreign antigen.
[0012] In one embodiment, the foreign antigen is a soluble
antigen.
[0013] In one embodiment, the first dose of the at least one
anti-CD4 antibody is administered prior to administration of the
foreign antigen.
[0014] In one embodiment, the at least one anti-CD4 antibody is
TRX1.
[0015] In another aspect, the invention is directed to a method for
treating a primate to induce tolerance to at least one foreign
antigen comprising, administering to the primate at least one
anti-CD4 antibody or CD4 binding fragment thereof, wherein at least
one dose of the at least one anti-CD4 antibody is administered at
least one day prior to administration of the foreign antigen.
[0016] In yet another aspect, the invention is directed to a method
for treating a primate to induce tolerance to at least one foreign
antigen comprising, administering to the primate at least one
anti-CD4 antibody or CD4 binding fragment thereof, wherein the at
least one anti-CD4 antibody is administered at a dose sufficient to
maintain a serum concentration of anti-CD4 antibody at a level of
about 20 .mu.g/ml during the tolerance induction phase.
[0017] In one embodiment, at least one dose of the at least one
anti-CD4 antibody is administered one day prior to administration
of the foreign antigen.
[0018] In another embodiment, the at least one anti-CD4 antibody is
administered at a dose of between about 20 mg/kg and 40 mg/kg.
[0019] In another aspect, the invention is directed to a method for
treating a primate to induce tolerance to at least one foreign
antigen comprising, administering to the primate at least one
anti-CD4 antibody or CD4 binding fragment thereof, wherein the at
least one anti-CD4 antibody is administered at a dose sufficient to
achieve about 85% saturation of CD4 sites on T cells in the primate
during the tolerance induction phase.
[0020] In one embodiment, the at least one anti-CD4 antibody is not
administered for more than about two weeks.
[0021] In one embodiment, at least one dose of the at least one
anti-CD4 antibody is administered one day prior to administration
of the foreign antigen.
[0022] In another embodiement, the at least one anti-CD4 antibody
is administered at a dose of between about 20 mg/kg and 40
mg/kg.
[0023] In one embodiment, the at least one anti-CD4 antibody is
TRX1.
[0024] In another aspect, the invention pertains to a method for
inducing tolerance in a primate to a soluble antigen, comprising,
administering to a primate at least one anti-CD4 antibody or CD4
binding fragment thereof, wherein the at least one anti-CD4
antibody is administered at a dose of between about 20-40 mg/kg in
at least three separate doses, such that tolerance to the soluble
antigen is induced.
[0025] In one embodiment, at least one dose of the at least one
anti-CD4 antibody is administered one day prior to administration
of the soluble antigen.
[0026] In another embodiment, the at least one anti-CD4 antibody is
administered at a dose of about 20 mg/kg.
Brief Description of the Drawings
[0027] FIG. 1A shows the amino acid sequence of the first
embodiment of TRX1 antibody light chain. FIG. 1B shows the
nucleotide sequence of the first embodiment of TRX1 antibody light
chain. FIG. 1C shows the amino acid sequence of the first
embodiment of TRX1 antibody light chain with and without a leader
sequence. FIG. 1D shows the amino acid sequence of the first
embodiment of TRX1 antibody heavy chain. FIG. 1E is a continuation
of the sequence shown in FIG. 1D. FIG. 1F shows the nucleotide
sequence of the first embodiment of TRX1 antibody heavy chain. FIG.
1G shows the amino acid sequence of the first embodiment of TRX1
antibody heavy chain with and without a leader sequence.
[0028] FIG. 2A shows the amino acid sequence of another embodiment
of TRX1 antibody light chain. FIG. 2B shows the nucleotide sequence
of another embodiment of TRX1 antibody light chain. FIG. 2C shows
the amino acid sequence of another embodiment of TRX1 antibody
light chain with and without a leader sequence. FIG. 2D shows the
amino acid sequence of another embodiment of TRX1 antibody heavy
chain. FIG. 2E is a continuation of the sequence shown in FIG. 2D.
FIG. 2F shows the nucleotide sequence of another embodiment of TRX1
antibody heavy chain. FIG. 2G shows the amino acid sequence of
another embodiment of TRX1 antibody heavy chain with and without a
leader sequence.
[0029] FIG. 3A shows the amino acid sequence of another embodiment
of TRX1 antibody light chain. FIG. 3B shows the nucleotide sequence
of another embodiment of TRX1 antibody light chain. FIG. 3C shows
the amino acid sequence of another embodiment of TRX1 antibody
light chain with and without a leader sequence. FIG. 3D shows the
amino acid sequence of another embodiment of TRX1 antibody heavy
chain. FIG. 3E is a continuation of the sequence shown in FIG. 3D.
FIG. 3F shows the nucleotide sequence of another embodiment of TRX1
antibody heavy chain. FIG. 3G shows the amino acid sequence of
another embodiment of TRX1 antibody heavy chain with and without a
leader sequence.
[0030] FIG. 4A shows the amino acid sequence of another embodiment
of TRX1 antibody light chain. FIG. 4B shows the nucleotide sequence
of another embodiment of TRX1 antibody light chain. FIG. 4C shows
the amino acid sequence of another embodiment of TRX1 antibody
light chain with and without a leader sequence. FIG. 4D shows the
amino acid sequence of another embodiment of TRX1 antibody heavy
chain. FIG. 4E is a continuation of the sequence shown in FIG. 4D.
FIG. 4F shows the nucleotide sequence of another embodiment of TRX1
antibody heavy chain. FIG. 4G shows the amino acid sequence of
another embodiment of TRX1 antibody heavy chain with and without a
leader sequence.
[0031] FIGS. 5A-5C show the sequence of the heavy chains of the
humanized CD8 antibody used in Example 5.
[0032] FIG. 6 shows the sequence of the light chains of the
humanized CD8 antibody used in Example 5.
[0033] FIGS. 7A-7C show another embodiment of a TRX1 heavy chain.
In this embodiment, the amino acid at position 236 is a Leu, the
amino acid at position 238 is a Gly, and the amino acid at position
297 is an Ala.
[0034] FIGS. 8A-8B show another embodiment of a TRX1 light chain.
In this embodiment, the amino acid at position 117 is a Leu.
[0035] FIGS. 9A-9B show a schematic overview of the tolerance
induction and antigen challenge protocol. 9A, The protocol was
divided into 3 phases: induction, washout, and challenge. During
the induction phase TRX1 antibody or saline was infused on days -1,
3 or 4, 8 and 12. Antigen (equine Ig or saline) was administered on
days 0, 3 and 8. The induction phase was followed by a washout
phase during which serum levels of TRX1 and equine Ig were
monitored until no longer detectable. The challenge phase was
initiated on day 68 by dosing all animals with equine Ig as well as
a neoantigen, SRBC. Additional equine Ig challenges were
administered on day 95 and day 130. 9B, Treatment groups consisted
of 4 experimental TRX1 dosing cohorts and 2 control groups. The
experimental groups received 4 infusions of TRX1 at 1, 10, 20, or
40 mg/kg and 3 doses of antigen. Control group I, antigen only,
received 4 infusions of saline and 3 doses of equine Ig. Control
group II, TRX1 only, was comprised of 2 cohorts with animals
receiving 4 infusions of TRX1 at 20 or 40 mg/kg. Animals in control
group II received 3 doses of saline rather than antigen. All
animals were challenged three times with antigen and received a
single immunization with SRBC at the time of the first equine Ig
challenge.
[0036] FIGS. 10A-10C show pharmacokinetics and pharmacodynamics of
TRX1 during the induction and washout phases. 10A, Group mean TRX1
serum concentrations (.mu.g/ml). Experimental and control group II
animals receiving equivalent TRX1 doses (20 and 40 mg/kg) are
combined. The arrow indicates dosing with TRX1. Open symbols
represent animals grouped according to the dose of TRX1 received, 1
mg/kg (n=3); 10 mg/kg (n=3); 20 mg/kg (n=4); or 40 mg/kg (n=6);
10B, Saturation of CD4 sites on CD3+ cells in peripheral blood as a
function of TRX1 dose during tolerance induction and washout
phases. TRX1-biotin staining of whole blood samples was used to
detect free CD4 sites. The mean MCF value for each group is
represented as a percent of the group mean MCF baseline value;
Control group I, antigen only, closed circles (n=3); open symbols
represent animals grouped according to the dose of TRX1 received, 1
mg/kg group (n=3); 10 mg/kg (n=3); 20 mg/kg; and 40 mg/kg (n=6);
10C, Total CD4.sup.+ T cells per ml of blood. Group mean absolute
CD4.sup.+ lymphocyte counts are calculated as a percentage of group
mean baseline values; Control group I, antigen only, closed circles
(n=3); open symbols represent animals grouped according to the dose
of TRX1 received, 1 mg/kg (n=3); 10 mg/kg (n=3); 20 mg/kg (n=4); 40
mg/kg (n=6).
[0037] FIGS. 11A-11C show immune response during induction and
first challenge. 11A, Group mean antibody titers generated against
equine Ig during the induction phase. Animals received 3 doses of
antivenin indicated by arrows. Titer against antivenin is defined
as the reciprocal of the dilution resulting in an OD value
equivalent to twice the OD value of a 1:25,000 dilution of a
positive control standard. Closed circles are control group I,
antigen only (n=3); open symbols represent the TRX1 experimental
dosing cohorts, 1 mg/kg TRX1 (n=3); 10 mg/kg TRX1 (n=3); 20 mg/kg
TRX1 (n=2); and 40 mg/kg TRX1 (n=3); 11B, Group mean antibody
titers generated against equine Ig after the first challenge given
on day 68 (arrow). Closed circles are control group I, antigen only
(n=3); gray symbols are control group II, TRX1 only cohorts, 20
mg/kg TRX1 (n=2); and 40 mg/kg (n=3); and open symbols represent
the TRX1 experimental dosing cohorts, 1 mg/kg (n=3); 10 mg/kg
(n=3); 20 mg/kg (n=3); and 40 mg/kg (n=3); 11C, Immune response to
the neo-antigen, SRBC, administered at the time of first challenge
on day 68 (arrow) and measured by hemolysis of SRBC. Group mean
antibody titers for control group I, closed circles, (n=3); control
group II cohorts, gray symbols, 20 mg/kg TRX1 (n=2); and 40 mg/kg
(n=3); and TRX1 experimental dosing cohorts, open symbols, 1 mg/kg
(n=3); 10 mg/kg (n=3); 20 mg/kg (n=3); and 40 mg/kg (n=3). Titer is
defined as the reciprocal of the highest dilution of serum that did
not cause obvious hemolysis.
[0038] FIGS. 12A-12B show immune response to equine Ig after
multiple challenges. 12A, Group mean antibody titers for control
group I, closed circles, (n=3); Control group II cohorts, gray
symbols, 20 mg/kg TRX1 (n=2) and 40 mg/kg (n=3); and the TRX1
experimental group cohorts, open symbols, 20 mg/kg TRX1 (n=2) and
40 mg/kg TRX1 (n=3). 12B, Antibody titers to equine Ig of
individual animals in the TRX1 experimental group 20 mg/kg (#16276
and #16096, open symbols, solid lines) and 40 mg/kg (#16178,
#16192, and #16286, closed symbols, solid lines) cohorts plotted
with the group mean antibody titers to equine Ig for control group
I (gray circles, solid line) and control group II, 20 mg/kg (open
triangle, dotted line, n=2) and 40 mg/kg (closed square, dotted
line, n=3) cohorts.
[0039] FIGS. 13A-13B show immune response to equine Ig with
modified TRX1 dosing. Closed circles represent saline control group
I, or antigen only (n=3); open triangles represent the 20 mg/kg
TRX1 treated experimental group (n=2). Titer is defined as the
reciprocal of the dilution of serum resulting in an OD value
equivalent to twice the OD value of a 1:25,000 dilution of positive
control serum. 13A, Group mean antibody titers generated against
equine Ig during the induction phase. Animals received 3 doses of
TRX1 indicated by arrows on day -1, 1 and 3. Equine Ig was
administered on days 0, 4 and 8. 13B, Group mean antibody titers
generated against equine Ig during the challenge phase. Animals
were challenged with 10 mg/kg antivenin s.c. on days 68 and 97 and
with 1 mg/kg equine Ig s.c. on day 133.
DETAILED DESCRIPTION OF THE INVENTION
[0040] The antigen(s) as to which tolerance is induced may be a
self antigen or a foreign antigen and in particular a foreign
antigen(s).
[0041] As used herein, the term "tolerize" or "tolerant" or
"tolerance" includes refractivity to activating receptor-mediated
stimulation. Such refractivity is generally antigen-specific and
persists after exposure to the tolerizing antigen has ceased. For
example, tolerance is characterized by lack of cytokine production,
e.g., IL-2 upon subsequent exposure to the tolerizing antigen.
Tolerance can occur to self antigens or to foreign antigens. In one
embodiment, the a tolerant primate does not produce an adverse
immune response to the antigen over a period of time after
treatment with a tolerizing agent is stopped even when subsequently
challenged with the antigen and/or when the antigen remains present
in the primate, but is capable of providing an immune response
against other antigens. In one embodiment, tolerance is induced in
the absence of a therapeutic level of a general
immunosuppressant.
[0042] For example, the foreign antigen may be one or more of the
following types of antigens: [0043] (i) a foreign antigen(s)
present on transplanted tissue or cells, including tissue or cells
present in an organ wherein the transplant may be allogeneic or
xenogeneic; [0044] (ii) a therapeutic agent (which also includes
therapeutic agents used for disease prevention) that produces an
immune response in a primate, which immune response diminishes the
ability of the agent to function as a therapeutic agent. Such
agents include, but are not limited to, delivery vehicles, such as
vectors used in gene therapy; active agents such as proteins
delivered to the primate (e.g., recombinant proteins such as
monoclonal antibodies, enzymes, clotting factors) and some small
molecule drugs or proteins produced from an agent delivered to the
primate, such as in gene therapy.
[0045] The foreign antigens against which tolerance is induced in
accordance with the present invention are not foreign antigens as
present in disease-causing bacteria, fungi, viruses, etc. that
infect a host, i.e., the term foreign antigen does not include a
foreign antigen as part of an organism that infects a primate and
causes a disease or disorder.
[0046] In one embodiment, the antigen is a soluble antigen.
[0047] The CD4 antibody or CD8 antibody in the case where a CD8
antibody is used, is preferably a monoclonal antibody (or fragment
thereof that retains the ability to bind to CD4 or CD8,
respectively). The antibody may be a human antibody or a non-human
antibody, with non-human antibodies including humanized antibodies,
chimeric antibodies, murine antibodies, etc.
[0048] The CD4 antibody or appropriate fragment thereof is
administered to a primate in an amount and for a time effective to
induce tolerance against a foreign or self antigen and preferably a
foreign antigen. Anti-primate CD4 antibodies are known in the art
as are methods of making such antibodies.
[0049] In one embodiment, the anti-CD4 antibody is administered
prior to exposure (or systemic exposure) of the subject to the
antigen to which tolerance is desired. In another embodiment, the
anti-CD4 antibody is administered simultaneously with the antigen
to which tolerance is desired.
[0050] In certain embodiments, in particular where tolerance to a
transplant (e.g., a cell or tissue transplant) is desired, it may
be desirable to additionally inhibit CD8+ cells. The compound that
inhibits CD8+ T cells inhibits the activity of CD8+ T cells, e.g.,
by reducing their number or by inhibiting their effector function.
In one embodiment, a compound that inhibits CD8+ T cells
specifically inhibits CD8+ T cells. In another embodiment, a
compound that inhibits CD8+ T cells does not significantly inhibit
or deplete Treg cells. Such a compound may be an antibody that does
or does not deplete CD8+ T cells. Anti-primate CD8 antibodies are
known in the art as are methods for making such antibodies. The
compound that inhibits CD8+ T-cells may be a compound (other than
an antibody) that inhibits such CD8+ T cells (such compound other
than an antibody may or may not deplete CD8+ T cells. Exemplary
non-antibody compounds include, e.g., beta-galactoside-binding
protein (Blaser et al. 1998. Eur J. Immunol. 28:2311).
[0051] In one embodiment, the compound that inhibits CD8+ T cells
is administered prior to administration of the anti-CD4 antibody.
In another embodiment, the compound that inhibits CD8+ T cells is
administered simultaneously with the anti-CD4 antibody. In another
embodiment, the compound that inhibits CD8+ T cells is administered
subsequent to administration of the anti-CD4 antibody.
[0052] As used herein, the term "regulatory T cell" includes T
cells which produce low levels of IL-2, IL-4, IL-5, and IL-12.
Regulatory T cells produce TNF.alpha., TGF.beta., IFN-.gamma., and
IL-10, albeit at lower levels than effector T cells. Although
TGF.beta. is the predominant cytokine produced by regulatory T
cells, the cytokine is produced at levels less than or equal to
that produced by Th1 or Th2 cells, e.g., an order of magnitude less
than in Th1 or Th2 cells. Regulatory T cells can be found in the
CD4+ CD25+ population of cells (see, e.g., Waldmann and Cobbold.
2001. Immunity. 14:399). Regulatory T cells actively suppress the
proliferation and cytokine production of Th1, Th2, or naive T cells
which have been stimulated in culture with an activating signal
(e.g., antigen and antigen presenting cells or with a signal that
mimics antigen in the context of MHC, e.g., anti-CD3 antibody, plus
anti-CD28 antibody).
[0053] Representative examples of compounds (other than antibodies)
that inhibit CD8+ T cells include: Rapamycin (sirolimus) and
CellCept.RTM. (mycophenolate mofetil). In one embodiment, a
compound such as cyclosporin is preferably not used because,
although it inhibits CD8+ T cells, such compound also inhibits Treg
cells (e.g., by depletion).
[0054] The present invention has particular applicability to
inducing tolerance in a primate with respect to a transplant.
Preferably such a primate is a human. The transplant may be
allogeneic or xenogeneic.
[0055] In accordance with a preferred embodiment, each of the CD4
antibody or appropriate fragment thereof and the CD8 inhibiting
compound is administered over a period of time in order to maintain
in the primate appropriate levels of such antibody or fragment and
compound over a period of time that is sufficient to induce
tolerance.
[0056] In one embodiment, at least one anti-CD4 antibody or CD4
binding fragment thereof, wherein the at least one anti-CD4
antibody is administered at a dose of about 20 mg/kg. In another
embodiment, the at least one anti-CD4 antibody is administered at a
dose of between about 20 and 40 mg/kg. In another embodiment, the
at least one anti-CD4 antibody is administered at a dose of at
least about 20 mg/kg. In one embodiment, at least three doses of
the antibody are administered. Each dose may be administered over
time, e.g., may be diluted and infused over a twenty four hour
period, or portions of the dose may be administered over the course
of a twenty four hour period, e.g., in separate inoculations.
[0057] In another embodiment, four separate doses of the anti-CD4
antibody are administered, e.g., the at least one anti-CD4 antibody
is administered on at least four separate days.
[0058] In another embodiment, five separate doses of the anti-CD4
antibody are administered, e.g., the at least one anti-CD4 antibody
is administered on at least five separate days.
[0059] In another embodiment, at least one dose of the at least one
anti-CD4 antibody is administered at least about 7 days prior to
administration of the foreign antigen. In another embodiment, at
least one dose of the at least one anti-CD4 antibody is
administered at least about 5 days prior to administration of the
foreign antigen. In another embodiment, at least one dose of the at
least one anti-CD4 antibody is administered at least about 4 days
prior to administration of the foreign antigen. In another
embodiment, at least one dose of the at least one anti-CD4 antibody
is administered at least about 3 days prior to administration of
the foreign antigen. In another embodiment, at least one dose of
the at least one anti-CD4 antibody is administered at least about 2
days prior to administration of the foreign antigen. In another
embodiment, at least one dose of the at least one anti-CD4 antibody
is administered at least about 1 day prior to administration of the
foreign antigen.
[0060] In one embodiment, administration of at least one anti-CD4
antibody continues for approximately one week after exposure to the
foreign antigen. In yet another embodiment, administration of at
least one anti-CD4 antibody continues for approximately two weeks
after exposure to the foreign antigen. In another embodiment,
administration of at least one anti-CD4 antibody continues for
approximately one month after exposure to the foreign antigen.
[0061] In another embodiment, the at least one anti-CD4 antibody is
administered on at least days -1, 3 or 4, 8 and 12 relative to
administration of the foreign antigen.
[0062] In yet another embodiment, the at least one anti-CD4
antibody is administered on at least days -1, 1, and 3 relative to
administration of the foreign antigen.
[0063] In another embodiment, the invention pertains to a process
for treating a primate to induce tolerance to at least one foreign
antigen comprising, administering to the primate at least one
anti-CD4 antibody or CD4 binding fragment thereof, wherein the at
least one anti-CD4 antibody is administered at a dose sufficient to
achieve slightly less than complete saturation of CD4 sites, e.g.,
about 95%, about 90%, about 85%, about 80%, or about 75% saturation
of CD4 sites on T cells in the primate during the tolerance
induction phase. In another embodiment, at least one anti-CD4
antibody is administered at a dose sufficient to achieve slightly
less than complete saturation of CD4 sites, e.g., about 95%, about
90%, about 85%, about 80%, or about 75% saturation of CD4 sites on
T cells in the primate during the tolerance induction phase. In one
embodiment, the level of saturation of CD4 sites does not exceed
about 85%. CD4 saturation can be determined using methods known in
the art. For example, in the appended examples, saturation was
determined as a function of free CD4 sites on circulating
lymphocytes. For example, free CD4 sites can be determined by
staining with anti-CD4, e.g., comprising a detectable label.
[0064] In one embodiment, administration of anti-CD4 antibody is
not necessary after the tolerance induction phase.
[0065] In still another embodiment, the invention pertains to a
process for inducing tolerance in a primate to a transplanted
antigen, comprising, administering to a primate at least one
anti-CD4 antibody or CD4 binding fragment thereof and at least one
compound that inhibits CD8+ T cells each in an amount and for a
time effective to induce tolerance against the transplant, said
anti-CD4 antibody or fragment being present in said primate when
said transplanted antigen is present in said primate and said
anti-CD4 antibody being administered in an initial dose of about 20
mg/kg, such that tolerance to the transplanted antigen is
induced.
[0066] In one embodiment, the dose of anti-CD4 may vary at each of
the times given during the induction phase. For example, the CD4
antibody (or fragment thereof) may be administered in an initial
dose, e.g. as set forth herein, and subsequent doses may be greater
than, equal to, or less than the initial dose.
[0067] Preferably, the dose of anti-CD4 administered (either the
initial or subsequent doses) is at least about 20 mg/kg. In another
embodiment, less than or equal to 40 mg/kg is administered in a
dose. In another embodiment, between about 20 mg/kg and 40 mg/kg is
administered in a dose. In yet another embodiment, between about 10
mg/kg and 40 mg/kg is administered in a dose. In yet another
embodiment, between about 5 and 40 mg/kg is administered in a
dose.
[0068] In one embodiment, the CD4 antibody (or fragment thereof)
may be administered in a dose of less than or equal to about 20
mg/kg. In another embodiment, the CD4 antibody (or fragment
thereof) may be administered in a dose of less than about 20 mg/kg.
For example, in instances when the anti-CD4 antibody is
administered to humans, it may be desirable to reduce the amount of
anti-CD4 antibody administered given the reduced immunogenicity and
improved pharmacokinetics of the TRX1 antibody in humans. For
example, in one embodiment, the dose of anti-CD4 administered
(either the initial or subsequent doses) is at least about 5 mg/kg.
In another embodiment, the dose of anti-CD4 administered (either
the initial or subsequent doses) is at least about 7.5 mg/kg. In
another embodiment, the dose of anti-CD4 administered (either the
initial or subsequent doses) is at least about 10 mg/kg. In another
embodiment, the dose of anti-CD4 administered (either the initial
or subsequent doses) is at least about 12.5 mg/kg. In another
embodiment, the dose of anti-CD4 administered (either the initial
or subsequent doses) is at least about 15 mg/kg. In another
embodiment, the dose of anti-CD4 administered (either the initial
or subsequent doses) is at least about 17.5 mg/kg.
[0069] The initial dose of the CD4 antibody may be administered in
one or more parts over a twenty-four hour period and preferably in
one dose over twenty-four hours.
[0070] As used herein in reference to a dosage amount, a dose is
the total amount of the CD4 antibody administered over a
twenty-four hour period, even if administered more than once in 24
hours.
[0071] As used herein, the term "tolerance induction phase"
includes the time during which anti-CD4 antibody is administered to
a primate to induce tolerance. For example, anti-CD4 antibody may
be administered in three to four doses over a short time period
(e.g., for about a month or less, such as about 10, about 13, about
15, about 20, about 25, or about 30 days) in close proximity to the
time of exposure to the foreign antigen.
[0072] In most cases, after the initial dose, the CD4 antibody (or
appropriate fragment thereof) is administered in one or more
follow-up doses over several day(s), with each follow-up dose being
administered in one or more doses in a twenty-four hour period. The
follow-up dose(s) is generally provided in an amount to return
serum levels of the CD4 antibody to those that were achieved by the
initial dose.
[0073] In a preferred embodiment, the minimum follow-up dose or
doses of the CD4 antibody is in an amount that is generally equal
to the amounts hereinabove described and may or may not be
identical to the dose given as the original or initial dose.
[0074] If there is more than one follow-up dose of the CD4
antibody, each such additional follow-up dose over a 24-hour period
may be the same or different than another follow-up dose.
[0075] The number of follow-up doses of the CD4 antibody will vary.
In one embodiment, there is at least one follow-up dose. In one
embodiment, the total number of doses does not exceed eight daily
doses.
[0076] In one embodiment, subsequent doses of anti-CD4 antibody are
administered approximately every 4-5 days. In another embodiment,
subsequent doses of anti-CD4 antibody are administered
approximately every 2-3 days. In another embodiment, subsequent
doses of anti-CD4 antibody are administered approximately every 1-2
days.
[0077] In one embodiment, the total period over which the CD4
antibody is administered does not exceed four weeks and more
preferably does not exceed three weeks. In many cases, tolerance
can be achieved by using an initial dose and one or more follow-up
doses over a period that does not exceed two weeks.
[0078] Although, in accordance with the present invention, initial
tolerance to an antigen(s) can be achieved in a primate in a
tolerance induction period of no more than about four weeks, in
some cases, periodic follow-up treatments with the CD4 antibody may
be administered in order to maintain tolerance.
[0079] In one embodiment, the invention pertains to a process for
treating a primate to induce tolerance to at least one foreign
antigen comprising, administering to the primate at least one
anti-CD4 antibody or CD4 binding fragment thereof, wherein the at
least one anti-CD4 antibody is administered at a dose sufficient to
maintain a serum concentration of anti-CD4 antibody at a level of
about 20 .mu.g/ml during the tolerance induction. In another
embodiment, the serum concentration is maintained at a level of at
least about 20 .mu.g/ml.
[0080] As hereinabove described, at least one CD4 antibody (or
appropriate fragment thereof) is delivered in an amount that is at
least sufficient to induce tolerance in a primate against an
antigen(s) and in a preferred embodiment against a foreign antigen.
The maximum amount is of course limited by safety considerations.
In general, the daily dosage of CD4 antibody would be less than
6000 mg.
[0081] The number of follow-up doses and the spacing thereof will
be determined, in part, by the half life of the at least one CD4
antibody. Although the present invention is not to be limited, in
one embodiment, the CD4 antibody will be initially delivered in an
amount to achieve antibody serum levels that exceed the amount
required to saturate all of the CD4 of the primate being treated,
with follow-up doses being given at times to maintain such excess
over a period that induces tolerance in the primate against the
foreign antigen(s).
[0082] In a preferred embodiment, the CD4 antibody is a CD4
antibody that would have a reduced effector (i.e. lytic) function
as compared to human IgG1. Examples of antibodies that would have
reduced effector function, include antibodies that have an Fc
portion that is aglycosylated and/or that has reduced binding to
the Fc receptor and/or is non-lytic. For example, in one
embodiment, an anti-CD4 antibody comprises at least one mutation in
the constant region of the heavy chain. Exemplary mutations include
Leu 236 to Ala (e.g., CTG to GCG), Gly 238 to Ala (e.g., GGA to
GCA), Asn 297 to Ala (e.g., AAC to GCC). In one embodiment, one or
more of these mutations may be made. For example, in a preferred
embodiment, the mutation at position 297 is made to produce an
aglycosyl anti-CD4 antibody with reduced effector function. In
another embodiment, the mutations at positions 236 and 238 are
made. This form is glycosylated, but Fc receptor and complement
binding are reduced.
[0083] In one embodiment, a CD4 antibody with a reduced effector
function is a non-depleting CD4 antibody. As used herein, "a
non-depleting CD4 antibody" is a CD4 antibody that depletes less
than 50% of CD4 cells and preferably less than 10% of CD4
cells.
[0084] In one embodiment, a cocktail comprising different anti-CD4
antibodies can be used. In another embodiment, different anti-CD4
antibodies can be administered to the same patient on different
days.
[0085] In one embodiment, a CD8 cell inhibiting compound is further
administered to enhance tolerance in a primate. The CD8 inhibiting
compound is administered to the primate during the initial
treatment with the CD4 antibody in an amount effective to reduce
the action and/or level of CD8+ T cells in the primate. Such
amounts may be lower than the amounts used for the CD4 antibody.
The CD8 inhibiting compound may be used at the same time as the CD4
antibody or may be used at different times. The CD8 inhibiting
compound may be administered on different days or on the same day
as the CD4 antibody. As hereinabove described, the CD8 inhibiting
compound may be an antibody (or fragment thereof) or a compound
other than an antibody. The treatment with the CD8 inhibiting
compound is performed during the initial treatment (including
initial follow-up doses); however, if further treatment with CD4
antibody is used after the initial treatment period (including
follow-up doses), such further treatment may be performed with or
without treatment with the CD8 inhibiting compound.
[0086] In treating a primate and in particular a human, the CD4
antibody (and optionally the CD8 inhibiting compound) may be
employed in combination with a pharmaceutically acceptable carrier,
e.g., formulated for separate or joint administration. A
composition that contains a CD4 antibody and/or CD8 inhibiting
compound may include other ingredients, for example, stabilizers
and/or other active agents.
[0087] The use of an anti-CD4 antibody to induce tolerance against
an antigen(s) in a primate in accordance with the present invention
provides tolerance against one or more antigens and the primate is
capable of immunologically responding to other antigens. Thus, in
this respect, the primate is made tolerant to one or more antigens,
and the immune system is capable of providing an immune response
against other foreign antigens whereby the primate is not
immunocompromised.
[0088] In the preferred embodiment where tolerance is induced
against a foreign antigen, each of the CD4 antibody (and optionally
the CD8 inhibiting compound, alone or in combination with each
other) is administered to the primate prior to, in conjunction with
or subsequent to the foreign antigen being delivered to the
primate. In a preferred embodiment, the primate is provided with
the CD4 antibody and the CD8 inhibiting compound at a time such
that both are present in the primate when the antigen(s) against
which tolerance is to be induced is also present in the primate. In
a particularly preferred embodiment, each of the CD4 antibody (or
fragment thereof) and the CD8 inhibiting compound is delivered to
the primate prior to the primate coming into contact with the
foreign antigen(s) to which the primate is to be tolerized or
within a few hours or less than one day thereafter.
[0089] In one embodiment, each of the CD4 antibody (and optionally
the CD8 inhibiting compound is administered to the primate at least
about 5, at least about 4, at least about 3, at least about 2, or
at least about 1 day prior to the primate receiving the foreign
antigen. In one embodiment, the anti-CD4 antibody is administered
about 5 days prior to the primate receiving the foreign
antigen.
[0090] As hereinabove indicated, in one embodiment, a primate is
tolerized against a therapeutic protein that is to be used in
treating the primate. Such therapeutic protein may be a soluble
antigen, e.g., a therapeutic antibody (other than the CD4 antibody)
(which therapeutic antibody may be a human antibody, humanized
antibody, chimeric antibody or a non-human antibody); an enzyme
such as one used for replacement therapy; a hormone; clotting
factor; a protein produced in gene therapy; a gene therapy delivery
vehicle such as a vector used in gene therapy (for example, an
adenovirus vector); or other soluble protein. As used herein, the
term "soluble" includes antigens which are not cell bound (such as
proteins which are naturally secreted by cells or which have been
engineered to be soluble, e.g., by removal of transmembrane and
cytoplasmic domains and/or by incorporation of various domains,
e.g., antibody Fc region domains.
[0091] The foreign antigen(s) may be present in a transplanted
organ, or in transplanted cells used in cell therapy, or in other
tissue transplants, such as skin.
[0092] In one embodiment, the primate has not been exposed to the
antigen prior to treatment with anti-CD4 antibody. In another
embodiment, the primate has been exposed to the antigen prior to
treatment with anti-CD4 antibody.
[0093] The treatment of a primate, in particular, a human, in order
to tolerize the primate against a foreign antigen by use of a CD4
antibody and a CD8 inhibiting compound may be accomplished in some
cases without adjunct therapy, such as a bone marrow transplant to
promote acceptance of a foreign antigen and/or
immunosuppression.
[0094] In some cases, adjunct therapy may also be employed. For
example, as part of a transplant procedure, immunosuppression with
an appropriate immunosuppressant may be used but by employing the
present invention, chronic immunosuppression is not required. In
addition, if used after or during the tolerizing procedure, in some
cases, the immunosuppressant may be used with less than the amount
required to provide for effective immunosuppression.
[0095] In one non-limiting embodiment, the CD4 antibody is
preferably a TRX1 antibody or one that binds to the same epitope as
TRX1, and such CD4 antibody is preferably used with the dosing
regimen as hereinabove described.
[0096] In accordance with an aspect of the present invention, such
CD4 antibody (preferably a humanized antibody or fragment thereof)
binds to the same epitope (or a portion thereof) on human
lymphocytes as the humanized antibody selected from the group
consisting of, the TRX1 humanized antibody, e.g., the components of
which, e.g., light chain and heavy chain, each containing constant
regions and variable regions, are depicted in FIGS. 1A-1G and
correspond to SEQ ID Nos.: 1, 2, 3, 4, 5, 6, 7 and 8; the TRX1
humanized antibody, e.g. the components of which, e.g., light chain
and heavy chain, each containing constant regions and variable
regions, are depicted in FIGS. 2A-2G and correspond to Seq ID Nos.:
9, 10, 11, 12, 13, 14, 15, and 16; the TRX1 humanized antibody,
e.g., the components of which, e.g., light chain and heavy chain,
each containing constant regions and variable regions, are depicted
in FIGS. 3A-3G and correspond to Seq ID Nos.: 17, 18, 19, 20, 21,
22, 23, and 24; and the TRX1 humanized antibody, e.g., the
components of which, e.g., light chain and heavy chain, each
containing constant regions and variable regions, are depicted in
FIGS. 4A-4G and correspond to Seq ID Nos.: 25, 26, 27, 28, 29, 30,
31, and 32.
[0097] The antibody is hereinafter sometimes referred to as TRX1.
The term "molecule" or "antibody that binds the same epitope as
TRX1" includes TRX1. The term "TRX1" includes the components of the
humanized antibody, e.g. light chain and heavy chain, each
containing a constant region and a variable region, e.g., amino
acid sequences shown in Seq ID Nos.: 1, 3, 4, 5, 7 and 8 (FIGS. 1A,
1C, 1D, 1E, and 1G), the components of the humanized antibody,
e.g., light chain and heavy chain, each containing a constant
region and a variable region, e.g., amino acid sequences shown in
Seq ID Nos.: 9, 11, 12, 13, 15, and 16 (FIGS. 2A, 2C, 2D, 2E, and
2G), the components of the humanized antibody, e.g., light chain
and heavy chain, each containing a constant region and a variable
region, e.g., amino acid sequences shown in Seq ID Nos.: 17, 19,
21, 23, and 24 (FIGS. 3A, 3C, 3D, 3E, and 3G), the components of
the humanized antibody, e.g., light chain and heavy chain, each
containing a constant region and a variable region, e.g., amino
acid sequences shown in Seq ID Nos.: 25, 27, 28, 29, 31, and 32
(FIGS. 4A, 4C, 4D, 4E, and 4G), and those identical thereto which
may be produced, for example, by recombinant technology.
[0098] Although the preferred CD4 antibody is TRX1, from the
teachings herein, other anti-CD4 antibodies can also be employed in
the methods of the invention. For example, in one embodiment, one
skilled in the art can produce antibodies that are equivalent to
TRX1. For example, such antibodies may be: [0099] 1) humanized
antibodies that bind to CD4 (e.g., by binding to the same epitope
as TRX1); [0100] 2) humanized antibodies that have the same CDRs as
TRX1 but which have a different humanized framework and/or a
different human constant region; [0101] 3) humanized antibodies
that bind to CD4 (e.g., by binding to the same epitope as TRX1) in
which one or more amino acids of one or more of the CDRs of TRX1
have been changed (preferably but not necessarily a conservative
amino acid substitution) and in which the framework may be the same
framework as TRX1 or have a different humanized framework or in
which one or more of the amino acids of the framework region of
TRX1 have been changed and/or in which the constant region may be
the same as or different from TRX1; [0102] 4) humanized antibodies
that bind to CD4 (e.g., by binding to the same epitope as TRX1)
wherein the antibody does not bind to Fc receptors through the Fc
region of the antibody. [0103] 5) humanized antibodies that bind to
CD4 (e.g., by binding to the same epitope as TRX1) wherein the CDRs
thereof do not include a glycosylation site; [0104] 6) humanized
antibodies that bind to CD4 (e.g., by binding to the same epitope
as TRX1) and that do not bind to Fc receptors through the Fc region
of the antibody and the CDRs do not include a glycosylation site;
[0105] 7) a chimeric antibody that bind to CD4 (e.g., by binding to
the same epitope as TRX1); and [0106] 8) a murine antibody that
bind to CD4 (e.g., by binding to the same epitope as TRX1).
[0107] The antibodies that are equivalent to TRX1 may be used in
the same manner and for the same purposes as TRX1.
[0108] In a preferred embodiment, the CD4 antibody employed in the
present invention is one which binds to the same epitope (or a part
of that epitope) as the TRX1 humanized antibody. The term "binds to
the same epitope as TRX1 humanized antibody" is intended to
describe not only the TRX1 humanized antibody but also describes
other antibodies, fragments or derivatives thereof that bind to the
same such epitope as the TRX1 humanized antibody. Antibodies that
bind to the same epitope as TRX1 humanized antibody can be
identified using techniques known to those of ordinary skill in the
art, e.g., antibody competition assays or epitope mapping.
[0109] In a preferred embodiment, the CD4 antibody does not bind to
Fc receptors through the Fc region of the antibody and the CDRs do
not include a glycosylation site.
[0110] The constant region may or may not include a glycosylation
site. In one embodiment, the constant region includes a
glycosylation site. Glycosylation signals are well known in the
art. An example of a heavy chain sequence which includes a
glycosylation site is shown in SEQ ID NO.:5 (FIGS. 1D and 1E), SEQ
ID NO.:7 (FIG. 1G) and SEQ ID NO.:8 (FIG. 1G), and SEQ ID NO.:21
(FIGS. 3D and 3E), SEQ ID NO.:23 (FIG. 3G) and SEQ ID NO.:24 (FIG.
3G). In another embodiment, the constant region does not include a
glycosylation site due to an asparagine (N) to an alanine (A) amino
acid change. An example of a heavy chain sequence which does not
include a glycosylation site is shown in SEQ ID NO.: 13 (FIGS. 2D
and 2E), SEQ ID NO.:15 (FIG. 2G) and SEQ ID NO.:16 (FIG. 2G), and
SEQ ID NO.: 29 (FIGS. 4D and 4E), SEQ ID NO.:31 (FIG. 4G) and SEQ
ID NO.:32 (FIG. 4G).
[0111] Such other antibodies include, by way of example and not by
limitation, rat, murine, porcine, bovine, human, chimeric,
humanized antibodies, or fragments or derivatives thereof.
[0112] The term "fragment" as used herein means a portion of an
antibody, by way of example, such portions of antibodies shall
include but not be limited to CDR, Fab, scFv molecules or such
other portions, which bind to the same epitope or any portion
thereof as recognized by TRX1.
[0113] The term "antibody" as used herein includes polyclonal and
monoclonal antibodies as well as antibody fragments and
derivatives, as well as antibodies prepared by recombinant
techniques, such as chimeric or humanized antibodies, single chain
or bispecific antibodies which bind to the same epitope or a
portion thereof as recognized by the humanized antibody TRX1. The
term "molecules" includes by way of example and not limitation,
peptides, oligonucleotides or other such compounds derived from any
source which mimic the antibody or binds to the same epitope or a
portion thereof as the antibody fragment or derivative thereof.
[0114] Another embodiment of the present invention provides for a
method of treating a patient who is to receive or has received a
graft transplant with an effective amount of (i) at least one
member selected from the group consisting of TRX1 antibody, or an
antibody, or derivative or fragment thereof that bind to the same
epitope (or a portion thereof) as the TRX1 antibody and (ii) a CD8
inhibiting compound. The treatment is preferably effected with the
whole or intact TRX1 antibody.
[0115] In one embodiment, the anti-CD4 antibody used in the methods
of the invention is humanized and is modified to reduce effector
function, e.g., by modification to reduce Fc receptor and/or
complement binding using methods known in the art.
[0116] In one embodiment, the antibody is TRX1 (SEQ ID Nos.:1, 2,
3, 4, 5, 6, 7, and 8; FIGS. 1A, 1B, 1C, 1D, 1E, 1F, and 1G). The
TRX1 antibody, e.g., the components of the TRX1 antibody, e.g., the
light chain and heavy chain, each containing variable and constant
regions, which are shown in, e.g., SEQ ID Nos.: 1 (FIG. 1A), 2,
(FIG. 1B), 3 (FIG. 1C, top), 4 (FIG. 1C, bottom), 5 (FIGS. 1D and
1E), 6 (FIG. 1F), 7 (FIG. 1G, top), and 8 (FIG. 1G, bottom). SEQ ID
No.:1 (FIG. 1A) is the amino acid sequence of the TRX1 light chain
and SEQ ID No.:2 (FIG. 1B) is the nucleotide sequence of the TRX1
light chain. SEQ ID No.:3 (FIG. 1C, top) is the amino acid sequence
of the TRX1 light chain, with a leader sequence. SEQ ID No.:4 (FIG.
1C, bottom) is the amino acid sequence of the TRX1 light chain,
e.g., SEQ ID No.:1 or SEQ ID No.:3, without a leader sequence,
e.g., amino acid residues 1-20 of SEQ ID No.:1. The TRX1 heavy
chain amino acid sequence, containing a glycosylation site, e.g.,
amino acid residues 317-319, is shown in SEQ ID No.:5 (FIGS. 1D and
1E) and the nucleotide sequence of the TRX1 heavy chain is shown in
SEQ ID No.:6 (FIG. 1F). SEQ ID No.:7 (FIG. 1G, top) is the amino
acid sequence of the TRX1 heavy chain with a leader sequence. SEQ
ID No.:8 (FIG. 1G, bottom) is the amino acid sequence of the TRX1
heavy chain, e.g., SEQ ID No.:5 (FIGS. 1D and 1E), without a leader
sequence, e.g., amino acid residues 1-19 of SEQ ID No.:5 (FIGS. 1D
and 1E), and contains a glycosylation site, e.g., amino acid
residues 298-300. TRX1 is a humanized antibody that includes
modified constant regions of a human antibody, e.g., light chain
amino acid residues 132-238 of SEQ ID No.:1 (FIG. 1A) or SEQ ID
No.:3 (FIG. 1C, top), and amino acid residues 112-218 of SEQ ID
No.:4 (FIG. 1C, bottom), and heavy chain amino acid residues
138-467 of SEQ ID No,:5 (FIGS. 1D and 1E) or SEQ ID No.:7 (FIG. 1G,
top) and amino acid residues 119-448 of SEQ ID No.:8 (FIG. 1G), and
light and heavy chain framework and CDR regions, in which the
framework regions of the light and heavy chain variable regions
correspond to the framework regions of the light chain variable
region, e.g., amino acid residues 21-43, 59-73, 81-112, and 122-131
of SEQ ID No.:1 (FIG. 1A) or SEQ ID No.:3 (FIG. 1C, top) and amino
acid residues 1-22, 33-53, 61-92, and 102-111 of SEQ ID No.:4 (FIG.
1C), and framework regions of the heavy chain variable region,
e.g., amino acid residues 20-49, 55-68, 86-117, and 127-137 of SEQ
ID No.:5 or SEQ ID No.:7 (FIG. 1G, top) and amino acid residues
1-30, 36-49, 67-98, and 108-118 of SEQ ID No.:8, which are derived
from a human antibody, and the CDRs of the light chain, e.g., amino
acid residues 44-58, 74-80, and 113-121 of SEQ ID No.:1 or SEQ ID
No.:3 (FIG. 1C, top), and amino acid residues 24-32, 54-60, and
93-101 of SEQ ID No.:4, and the CDRs of the heavy chain, e.g.,
amino acid residues 50-54, 69-85, and 118-126 of SEQ ID No.:5 or
SEQ ID No.:7 (FIG. 1G, top) and amino acid residues 31-35, 50-66,
and 99-107 of SEQ ID No.:8, which are derived from a mouse
monoclonal antibody designated NSM4.7.2.4.
[0117] In another embodiment, the antibody is TRX1 (SEQ ID Nos.:17,
18, 19, 20, 21, 22, 23, and 24; FIGS. 3A, 3B, 3C, 3D, 3E, 3F, and
3G). The TRX1 antibody, e.g., the components of the TRX1 antibody,
e.g., the light chain and heavy chain, each containing variable and
constant regions, are shown in, e.g., SEQ ID Nos.: 17 (FIG. 3A),
18, (FIG. 3B), 19 (FIG. 3C, top), 20 (FIG. 3C, bottom), 21 (FIGS.
3D and 3E), 22, (FIG. 3F) 23 (FIG. 3G, top), and 24 (FIG. 3G,
bottom). SEQ ID No.:17 (FIG. 3A) is the amino acid sequence of the
TRX1 light chain and SEQ ID No.:18 (FIG. 3B) is the nucleotide
sequence of the TRX1 light chain. SEQ ID No.:19 (FIG. 3C, top) is
the amino acid sequence of the TRX1 light chain with a leader
sequence. SEQ ID No.:20 (FIG. 3C, bottom) is the amino acid
sequence of the TRX1 light chain, e.g., SEQ ID No.:17, without a
leader sequence, e.g., amino acid residues 1-20 of SEQ ID No.:17.
The TRX1 heavy chain amino acid sequence, containing a
glycosylation site, e.g., amino acid residues 317-319 of SEQ ID
No.:21 (FIGS. 3D and 3E) and the nucleotide sequence of the TRX1
heavy chain is shown in SEQ ID No.:22 (FIG. 3F). SEQ ID No.:23
(FIG. 3G, top) is the amino acid sequence of the TRX1 heavy chain
with a leader sequence. SEQ ID No.:24 (FIG. 3G, bottom) is the
amino acid sequence of the TRX1 heavy chain, e.g., SEQ ID No.:21,
without a leader sequence, e.g., amino acid residues 1-19 of SEQ ID
No.:21, and contains a glycosylation site, e.g., amino acid
residues 298-300. TRX1 is a humanized antibody that includes
modified constant regions of a human antibody, e.g., light chain
amino acid residues 132-238 of SEQ ID No.:17 (FIG. 3A) or SEQ ID
No.:19 (FIG. 3C, top), and amino acid residues 112-218 of SEQ ID
No.:20 (FIG. 3C, bottom), and heavy chain amino acid residues
138-467 of SEQ ID No.:21 (FIGS. 3D and 3E) or SEQ ID No.:23 (FIG.
3G, top) and amino acid residues 119-448 of SEQ ID No.:24 (FIG. 3G,
bottom), and light and heavy chain framework and CDR regions, in
which the framework regions of the light and heavy chain variable
regions correspond to the framework regions of the light chain
variable region, e.g., amino acid residues 21-43, 59-73, 81-112,
and 122-131 of SEQ ID No.:17 (FIG. 3A) or SEQ ID No.:19 (FIG. 3C,
top), and amino acid residues 1-22, 33-53, 61-92, and 102-111 of
SEQ ID No.:20, and framework regions of the heavy chain variable
region, e.g., amino acid residues 2049, 55-68, 86-117, and 127-137
of SEQ ID No.:21 (FIGS. 3D and 3E) or SEQ ID No.:23 (FIG. 3G, top)
and amino acid residues 1-30, 36-49, 67-98, and 108-118 of SEQ ID
No.:24 (FIG. 3G, bottom), which are derived from a human antibody,
and the CDRs of the light chain, e.g., amino acid residues 44-58,
74-80, and 113-121 of SEQ ID No.:17 (FIG. 3A) or SEQ ID No.:19
(FIG. 3C, top), and amino acid residues 24-32, 54-60, and 93-101 of
SEQ ID No.:20 (FIG. 3C, bottom), and the CDRs of the heavy chain,
e.g., amino acid residues 50-54, 69-85, and 118-126 of SEQ ID
No.:21 (FIGS. 3D and 3E) or SEQ ID No.:23 (FIG. 3G, top) and amino
acid residues 31-35, 50-66, and 99-107 of SEQ ID No.:24 (FIG. 3G,
bottom), which are derived from a mouse monoclonal antibody
designated NSM4.7.2.4.
[0118] In another embodiment, the antibody is TRX1 (SEQ ID Nos.:9,
10, 11, 12, 13, 14, 15, and 16; FIGS. 2A, 2B, 2C, 2D, 2E, 2F, and
2G). The TRX1 antibody, e.g., the components of the TRX1 antibody,
e.g. the light chain and heavy chain, each containing variable and
constant regions, are shown in, e.g. SEQ ID Nos.: 9 (FIG. 2A), 10,
(FIG. 2B), 11 (FIG. 2C, top), 12 (FIG. 2C, bottom), 13 (FIGS. 2D
and 2E), 14 (FIG. 2F), 15 (FIG. 2G, top) and 16 (FIG. 2G, bottom).
SEQ ID No.:9 (FIG. 2A) is the amino acid sequence of the TRX1 light
chain and SEQ ID No.:10 (FIG. 2B) is the nucleotide sequence of the
TRX1 light chain. SEQ ID No.:11 (FIG. 2C) is the amino acid
sequence of the TRX1 light chain with a leader sequence. SEQ ID
No.:12 (FIG. 2C) is the amino acid sequence of the TRX1 light
chain, e.g., SEQ ID No.:9 (FIG. 2A), without a leader sequence,
e.g. amino acid residues 1-20 of SEQ ID No.:9. The TRX1 heavy chain
amino acid sequence, which does not contain a glycosylation site,
e.g., contains an asparagine to alanine change at amino acid
residue 317, is shown in SEQ ID No.:13 (FIGS. 2D and 2E) and the
nucleotide sequence of the TRX1 heavy chain is shown in SEQ ID
No.:14 (FIG. 2F). SEQ ID No.:15 (FIG. 2G, top) is the amino acid
sequence of the TRX1 heavy chain with a leader sequence. SEQ ID
No.:16 (FIG. 2G, bottom) is the amino acid sequence of the TRX1
heavy chain, e.g., SEQ ID No.:13, without a leader sequence, e.g.,
amino acid residues 1-19 of SEQ ID No.:13, and does not contain a
glycosylation site, e.g., contains an asparagine to alanine change
at amino acid residue 298. TRX1 is a humanized antibody that
includes modified constant regions of a human antibody, e.g., light
chain amino acid residues 132-238 of SEQ ID No.:9 (FIG. 2A) or SEQ
ID No.:11 (FIG. 2C, top), and amino acid residues 112-218 of SEQ ID
No.:12 (FIG. 2C, bottom), and heavy chain amino acid residues
138-467 of SEQ ID No.:13 (FIGS. 2D and 2E) or SEQ ID No.:15 (FIG.
2G, top) and amino acid residues 119-448 of SEQ ID No.:16 (FIG. 2G,
bottom), and light and heavy chain framework and CDR regions, in
which the framework regions of the light and heavy chain variable
regions correspond to the framework regions of the light chain
variable region, e.g., amino acid residues 21-43, 59-73, 81-112,
and 122-131 of SEQ ID No.:9 (FIG. 2A) or SEQ ID No.:11 (FIG. 2C,
top), and amino acid residues 1-22, 33-53, 61-92, and 102-111 of
SEQ ID No.:12 (FIG. 2C, bottom), and framework regions of the heavy
chain variable region, e.g., amino acid residues 20-49, 55-68,
86-117, and 127-137 of SEQ ID No.:13 (FIGS. 2D and 2E) or SEQ ID
No.:15 (FIG. 2G, top) and amino acid residues 1-30, 36-49, 67-98,
and 108-118 of SEQ ID No.:16 (FIG. 2G, bottom), which are derived
from a human antibody, and the CDRs of the light chain, e.g., amino
acid residues 44-58, 74-80, and 113-121 of SEQ ID No.:9 (FIG. 2A)
or SEQ ID No.:11 (FIG. 2C, top), and amino acid residues 24-32,
54-60, and 93-101 of SEQ ID No.:12 (FIG. 2C, bottom), and the CDRs
of the heavy chain, e.g., amino acid residues 50-54, 69-85, and
118-126 of SEQ ID No.:13 (FIGS. 2D and 2E) or SEQ ID No.:15 (FIG.
2G, top) and amino acid residues 31-35, 50-66, and 99-107 of SEQ ID
No.:16 (FIG. 2G, bottom), which are derived from a mouse monoclonal
antibody designated NSM4.7.2.4.
[0119] In another embodiment, the antibody is TRX1 (SEQ ID Nos.:25,
26, 27, 28, 29, 30, 31, and 32; FIGS. 4A, 4B, 4C, 4D, 4E, 4F, and
4G). The TRX1 antibody, e.g., the components of the TRX1 antibody,
e.g., the light chain and heavy chain, each containing variable and
constant regions, are shown in, e.g., SEQ ID Nos.: 25 (FIG. 4A), 26
(FIG. 4B), 27 (FIG. 4C, top), 28 (FIG. 4C, bottom), 29 (FIGS. 4D
and 4E), 30 (FIG. 4F), 31 (FIG. 4G, top), and 32 (FIG. 4G, bottom).
SEQ ID No.:25 (FIG. 4A) is the amino acid sequence of the TRX1
light chain and SEQ ID No.:26 (FIG. 4B) is the nucleotide sequence
of the TRX1 light chain. SEQ ID No.:27 (FIG. 4C, top) is the amino
acid sequence of the TRX1 light chain with a leader sequence. SEQ
ID No.:28 (FIG. 4C, bottom) is the amino acid sequence of the TRX1
light chain, e.g., SEQ ID No.:25, without a leader sequence, e.g.,
amino acid residues 1-20 of SEQ ID No.:25. The TRX1 heavy chain
amino acid sequence, which does not contain a glycosylation site,
e.g., contains an asparagine to alanine change at amino acid
residue 317, is shown in SEQ ID No.:29 (FIGS. 4D and 4E) and the
nucleotide sequence of the TRX1 heavy chain is shown in SEQ ID
No.:30 (FIG. 4F). SEQ ID No.:31 (FIG. 4G, top) is the amino acid
sequence of the TRX1 heavy chain with a leader sequence. SEQ ID
No.:32 (FIG. 4G, bottom) is the amino acid sequence of the TRX1
heavy chain, e.g., SEQ ID No.:29, without a leader sequence, e.g.,
amino acid residues 1-19 of SEQ ID No.:29, and does not contain a
glycosylation site, e.g., contains an asparagine to alanine change
at amino acid residue 298. TRX1 is a humanized antibody that
includes modified constant regions of a human antibody, e.g., light
chain amino acid residues 132-238 of SEQ ID No.:25 (FIG. 4A) or SEQ
ID No.:27 (FIG. 4C, top), and amino acid residues 112-218 of SEQ ID
No.:28 (FIG. 4C, bottom), and heavy chain amino acid residues
138-467 of SEQ ID No.:29 (FIGS. 4D and 4E) or SEQ ID No.:31 (FIG.
4G, top) and amino acid residues 119-448 of SEQ ID No.:32 (FIG. 4G,
bottom), and light and heavy chain framework and CDR regions, in
which the framework regions of the light and heavy chain variable
regions correspond to the framework regions of the light chain
variable region, e.g., amino acid residues 21-43, 59-73, 81-112,
and 122-131 of SEQ ID No.:25 (FIG. 4A) or SEQ ID No.:27 (FIG. 4C,
top), and amino acid residues 1-22, 33-53, 61-92, and 102-111 of
SEQ ID No.:28 (FIG. 4C, bottom), and framework regions of the heavy
chain variable region, e.g., amino acid residues 20-49, 55-68,
86-117, and 127-137 of SEQ ID No.:29 (FIGS. 4D and 4E) or SEQ ID
No.:31 (FIG. 4G, top) and amino acid residues 1-30, 36-49, 67-98,
and 108-118 of SEQ ID No.:32 (FIG. 4G, bottom), which are derived
from a human antibody, and the CDRs of the light chain, e.g., amino
acid residues 44-58, 74-80, and 113-121 of SEQ ID No.:25 (FIG. 4A)
or SEQ ID No.:27 (FIG. 4C, top), and amino acid residues 24-32,
54-60, and 93-101 of SEQ ID No.:28 (FIG. 4C, bottom), and the CDRs
of the heavy chain, e.g., amino acid residues 50-54, 69-85, and
118-126 of SEQ ID No.:29 (FIGS. 4D and 4E) or SEQ ID No.:31 (FIG.
4G, top) and amino acid residues 31-35, 50-66, and 99-107 of SEQ ID
No.:32 (FIG. 4G, bottom) which are derived from a mouse monoclonal
antibody designated NSM4.7.2.4.
[0120] In another embodiment, the TRX1 antibody comprises the heavy
chain sequence shown in FIGS. 7A-7C (SEQ ID NO:71 and 72). In
another embodiment, the TRX1 antibody comprises the heavy chain
sequence shown in FIGS. 7A-7C absent the leader sequence. In still
another embodiment, the TRX1 antibody comprises the light chain
sequence shown in FIGS. 8A-8B (SEQ ID NO:73 and 74). In still
another embodiment, the TRX1 antibody comprises the light chain
sequence shown in FIGS. 8A-8B absent the leader sequence.
[0121] In one embodiment, the invention provides an anti-CD4
antibody with a light chain variable region (LCVR) having at least
one CDR domain derived from a mouse monoclonal antibody, e.g.,
NSM4.7.2.4. In another embodiment, a light chain variable region
(LCVR) has at least one CDR domain comprising an amino acid
sequence selected from the group consisting of amino acid residues
44-58, 74-80, and 113-121 of, for example, SEQ ID No.:1 or SEQ ID
No.:3 or amino acid residues 24-32, 54-60, and 93-101 of SEQ ID
No.:4. In another embodiment, a light chain variable region (LCVR)
has at least two CDR domains comprising an amino acid sequence
selected from the group consisting of amino acid residues 44-58,
74-80, and 113-121 of, for example, SEQ ID No.:1 or SEQ ID No.:3 or
amino acid residues 24-32, 54-60, and 93-101 of SEQ ID No.:4. In
yet another embodiment, a light chain variable region (LCVR) has
CDR domains comprising the amino acid sequences consisting of amino
acid residues 44-58, 74-80, and 113-121 of, for example, SEQ ID
No.:1 or SEQ ID No.:3 or amino acid residues 24-32, 54-60, and
93-101 of SEQ ID No.:4.
[0122] In one embodiment of the invention, the anti-CD4 antibody
comprises a human framework region and a variable region comprising
at least one CDR derived from a mouse monoclonal antibody, e.g.,
NSM4.7.2.4. For example, in one embodiment, an anti-CD4 antibody
for use in the methods of the invention comprises at least one
light chain CDR sequence selected from the group consisting of
amino acid residues 44-58, 74-80, and 113-121 of, for example, SEQ
ID No.:1 or SEQ ID No.:3 or amino acid residues 24-32, 54-60, and
93-101 of SEQ ID No.:4. In one embodiment, an antibody for use in
the methods of the invention comprises at least two of the light
chain CDR sequences. In yet another embodiment, an antibody for use
in the methods of the invention comprises at least three of the
light chain CDR sequences.
[0123] In another embodiment, an anti-CD4 antibody for use in the
methods of the invention comprises at least one heavy chain CDR
sequence selected from the group consisting of amino acid residues
50-54, 69-85, and 118-126 of, for example, SEQ ID No.:5 or SEQ ID
No.:7 or amino acid residues 31-35, 50-66, and 99-107 of SEQ ID
No.:8. In one embodiment, an antibody for use in the methods of the
invention comprises at least two of the heavy chain CDR sequences.
In yet another embodiment, an antibody for use in the methods of
the invention comprises at least three of the heavy chain CDR
sequences.
[0124] Appropriate methods of preparing TRX1 humanized antibody or
other anti-CD4 antibody suitable for the purposes of the present
invention should be apparent to those skilled in the art from the
teachings herein. Such antibody may be prepared by recombinant
techniques known to those skilled in the art.
[0125] This invention is further illustrated by the following
examples, which should not be construed as limiting. The contents
of all references, patents and published patent applications cited
throughout this application, as well as the Figures, are
incorporated herein by reference.
EXAMPLES
[0126] The invention now will be described with respect to the
following examples; however, the scope of the present invention is
not intended to be limited thereby.
Example 1
Construction of TRX1 Antibody Starting from Amino Acid Sequence
[0127] A cDNA library was constructed from the mouse hybridoma NSM
4.7.2.4 using the Superscript plasmid system (Gibco/BRL, cat. no.
82485A) according to the manufacturer's suggested protocol. Heavy
and light chain cDNAs were cloned from the library by DNA
hybridization using as probes rat heavy and light chain gene cDNAs
from the rat hybridoma YTS 177.
[0128] The rat heavy and light chain gene cDNAs of YTS 177 were
isolated from the expression vector pHA Pr-1 as BamH1/Sal 1
fragments and labeled with .sup.32P and used independently to
screen the NSM 4.7.2.4. cDNA library using standard molecular
biology techniques (Sambrook, et al., Molecular Cloning, A.
Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. (2001); Ausubel, et al., Current
Protocols in Molecular Biology, John Wiley & Sons, New York
(2001).) Sequence analysis of the cDNAs derived from the NSM
4.7.2.4 cDNA library confirmed the NSM 4.7.2.4 heavy chain to be
mouse gamma-1 subclass and the NSM 4.7.2.4 light chain to be kappa.
The NSM 4.7.2.4 heavy and light V regions (VH and VL, respectively)
were reshaped to the human VH and VL regions with the "best fit" or
highest sequence similarity in the framework regions to that of the
mouse. For the light chain, human antibody HSIGKAW (from EMBL) with
a sequence similarity of 79% was used (L A Spatz et al., 1990 J.
Immunol. 144:2821-8). The sequence of HSIGKAW VL (SEQ ID No.35) is:
TABLE-US-00001 MVLQTQVFISLLLWISGAYGDIVMTQSPDSLAVSLGERATINCKSSQSLL
YSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLT
ISSLQAEDVAVYYCQQYYSTPPMFGQGTKVEIKRT
[0129] D start of framework 1 [0130] Q changed to G
[0131] For the heavy chain, human antibody A32483 (From GenBank)
with a sequence similarity of 74% was used (Larrick, et al.,
Biochem. Biophys. Res. Comm., Vol. 160, pgs. 1250-1256 (1989)). The
sequence of A32483 VH (SEQ ID No.36) is: TABLE-US-00002
LLAVAPGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQA
PGQGLEWMGIINPSGNSTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSED
TAVYYCAREKLATTIFGVLIITGMDYWGQGTLVTVSSGSAS
[0132] Q start of framework 1
[0133] For the humanization process, anti-CD4 light chain clone
77.53.1.2 (insert size 1 kb) and anti-CD4 heavy chain clone 58.59.1
(insert size 1.7 kb) were chosen from the cDNA library and inserts
isolated from the pSport vector as Sal I/Not I fragments and cloned
into M13mp18 vector to produce single stranded DNA for sequencing
and template for mutagenesis. The humanization of NSM 4.7.2.4 was
performed by site-directed mutagenesis of the mouse cDNA using a
kit from Amersham International (RPN 1523) according to the
manufacturer's suggested protocol.
[0134] Mutagenesis of the VL gene framework regions was performed
using five oligonucleotides ranging in length from 29 to 76 bases.
The oligos used were: TABLE-US-00003 Primer #1998 76 bases (SEQ ID
No.37) 5'-TGA CAT TGT GAT GAG CCA ATC TCC AGA TTC TTT GGC TGT GTC
TCT AGG TGA GAG GGC CAC CAT CAA CTG CAA GGC C Primer #1999 29 bases
(SEQ ID No.38) 5'-TGA ACT GGT ATC AAC AGA AAC CAG GAG AG Primer
#2000 28 bases (SEQ ID No.39) 5'-AGA GTC TGG GGT CCC AGA CAG GTT
TAG T Primer #2001 42 bases (SEQ ID No.40) 5'-GTC TTC AGG ACC CTC
CGA CGT TCG GTG GAG GTA CCA AGC TGG Primer #2008 52 bases (SEQ ID
No.41) 5'-CAC CCT CAC CAT GAG TTC TCT GCA GGC GGA GGA TGT TGC AGT
CTA TTA GTG T
[0135] The oligos were phosphorylated and mutagenesis performed in
three steps using no more than two oligos per step to introduce
changes according to the following procedure: [0136] (1) Annealing
phosphorylated mutant oligos to ssDNA template [0137] (2)
Polymerization [0138] (3) Filtration to remove single-stranded DNA
[0139] (4) Nicking non mutant strand with Nci I [0140] (5)
Digestion of non-mutant strand with Exo III [0141] (6)
Repolymerization of gapped DNA [0142] (7) Transformation of
competent JM101 [0143] (8) Sequencing of clones
[0144] Mutations were confirmed by single strand DNA sequencing
using M13 primers -20 and -40 and also the mutagenic primers # 1999
and # 2000.
[0145] A Sal I site at the 5' end of the variable region was
changed to Hind III by linker oligos #2334 and #2335 to allow
cloning of the variable region as a Hind III/Kpn I fragment into
the light chain constant region of CAMPATH-1H. TABLE-US-00004
Primer #2334 24 bases (SEQ ID No.42) 5'-AGC TTT ACA GTT ACT GAG CAC
ACA Primer #2335 24 bases (SEQ ID No.43) 5'-TCG ATG TGT GCT CAG TAA
CTG TAA
[0146] Mutagenesis of the VH gene framework regions was performed
using five oligonucleotides ranging in length from 24 to 75 bases.
The oligos used were: TABLE-US-00005 Primer #2003 75 bases (SEQ ID
No.44) 5'-GGT TCA GCT GGT GCA GTC TGG AGC TGA AGT GAA GAA GCC TGG
GGC TTC AGT GAA GGT GTC CTG TAA GGC TTC TGG Primer #2004 52 bases
(SEQ ID No.45) 5'-AGC TGG GTG AGG CAG GCA CCT GGA CAG GGC CTT GAG
TGG ATG GGA GAG ATT T Primer #2005 60 bases (SEQ ID No.46) 5'-CAA
GGG CAG GGT CAC AAT GAC TAG AGA CAC ATC CAC CAG CAC AGT CTA CAT GGA
ACT CAG Primer #2006 43 bases (SEQ ID No.47) 5'-CAG CCT GAG GTC TGA
GGA CAC TGC GGT CTA TTA CTG TGC AAG A Primer #2007 24 bases (SEQ ID
No.48) 5'-GCC AAG GGA CACG TAG TCA CTG TGT
[0147] Mutagenesis was carried out as described above for the light
chain again using no more than two oligos at a time to introduce
the changes. Mutations were confirmed by single strand DNA
sequencing using M13 primers -20 and -40 as well as the mutagenic
primers #2002 and #2004.
[0148] Primer #2002 was used to correct a reading frame error in
starting clone 58.59.1. TABLE-US-00006 Primer #2002 39 bases (SEQ
ID No.49) 5'-ACT CTA ACC ATG GAA TGG ATC TGG ATC TTT CTC CTC
ATC
[0149] Primer #2380 was used to correct extra mutation added by
#2004 which was missed in the first sequencing. TABLE-US-00007
Primer #2380 39 bases (SEQ ID No.50) 5'-TCA CTG CCT ATG TTA TAA GCT
GGG TGA GGC AGG CAC CTG
[0150] As with the light chain, the heavy chain 5' Sal I site was
changed to Hind III using linker oligo's #2334 and #2335 to allow
cloning of the heavy chain variable region as Hind III/Spe I (site
introduced by primer #2007) fragment into the heavy chain constant
region of CAMPATH-1H.
[0151] Construction of Heavy Chain
[0152] The following samples of DNA were used:
[0153] 1. Plasmid 1990. Human gamma-1 heavy chain constant region
gene cloned into pUC18 (obtained from Martin Sims, Wellcome
Foundation Ltd).
[0154] 2. Plasmid 2387: Reshaped heavy chain of NSM 4.7.2.4
containing human framework regions and mouse gamma 1 constant
region.
[0155] A Sal I site in the reshaped CD4 heavy chain was altered to
a Hind III site. The variable region gene was excised by digestion
with Hind III/Spe I and ligated with the constant region gene in
plasmid 1990 to give a complete humanized heavy chain (plasmid
2486). The heavy chain gene was cut out of this plasmid with Hind
III/EcoR I and ligated with the expression vector pEE6.
[0156] Construction of Light Chain
[0157] The following samples of DNA were used.
[0158] 1. Plasmid 2028; CAMPATH-1H light chain gene cloned into
M13mp18 at Sal I/BamH I restriction site.
[0159] 2. Plasmid 2197; Reshaped light chain of NSM 4.7.2.4
containing human framework regions and mouse kappa constant region.
A Kpn I site already had been introduced between variable and
constant portions of this gene.
[0160] A Kpn I restriction site was introduced into the CAMPATH 1H
light chain gene corresponding to the site in plasmid 2197 and an
EcoR I site was introduced at the 3' end of the constant region.
The constant region gene was excised from this plasmid (2502) by
digestion with Hind III/Kpn I.
[0161] Meanwhile a Sal I site in plasmid 2197 was changed to a Hind
III site (this step had to be repeated because a frame-shift
mutation inadvertently was introduced the first time). The new
plasmid (2736) was digested with Hind III/Kpn I. The CD4 variable
region fragment was cloned into a plasmid containing the kappa
constant region gene from plasmid 2502 to give a complete humanized
light chain (plasmid 2548). The light chain gene was cut out from
this plasmid with Hind III/EcoR I and ligated with the expression
vector pEE12 to give plasmid 2798.
[0162] Ligation of Heavy and Light Chains and Expression in NSO
Cells
[0163] The heavy chain gene was excised from the pEE6 vector by
digestion with Sal I/Bgl H and cloned into the light chain pEE12
vector which had been digested with BamH I/Sal I.
[0164] The final vector construct was checked by restriction
digests with Hind III, EcoR I, Sal I, BamH I, BgI II and Spe I for
the presence of the expected fragments, including 700 bp light
chain, 1400 bp heavy chain, 2300 bp fragment of pEE6 and 7000 bp
fragment of pEE12.
[0165] The pEE12 vector was linearized by digestion with Sal I and
transferred into NSO cells by electroporation, following a standard
protocol (Celltech 1991) except that the selection medium was
slightly modified, being based on IMDM rather than DMEM.
Transfectants were selected in medium lacking glutamine,
supplemented with dialysed FCS, ribonucleosides, glutamic acid, and
asparagine as recommended.
[0166] The transfection mixes were cultured in three 96-well
plates, and of 36 growing wells which were tested, 5 were strongly
positive for production of human heavy and light chains (18 others
were positive for one or other, or weakly positive for both).
[0167] A clone, designated SDG/B7B.A.7 was selected and stored
frozen but no further characterization has been done on this wild
type antibody.
[0168] Construction of Mutant IgG1 Antibody Designated to Abolish
Effector Functions
[0169] Due to concerns about side effects of other CD4 antibodies
reported in various clinical trials, it is considered desirable to
avoid the possibility of engaging Fc receptors. Human IgG4 is
thought to have minimal Fc binding or complement-activating
ability. However, experiments have show that it does engage Fc
receptors in some individuals (Greenwood et al., Eur. J. Immunol.,
Vol. 23, pgs. 1098-1104, 1993), and clinical studies with a human
IgG4 variant to CAMPATH-1H have demonstrated an ability to kill
cells in vivo (Isaacs et al., Clin. Exp. Immunol. Vol. 106, pgs.
427-433 (1996)). To eliminate the possibility of binding Fc
receptors, constructs were made with mutations in the IgG1 heavy
chain constant region.
[0170] TRX1 can be made to have mutations, e.g., Leu.sup.236 to Ala
and Gly.sup.238 to Ala, as shown in SEQ ID Nos.:5 and 6, and SEQ ID
Nos.:21 and 22. These particular residues were chosen because they
are predicted to disrupt maximally binding to all three types of
human Fc receptors for IgG. Either mutation is sufficient to reduce
binding to Fc(RI (Woof, et al., Mol. Immunol, Vol. 332, pgs.
563-564, 1986; Duncan, et al., Nature, Vol. 332, pgs. 563-564 1988;
Lund, et al., J. Immunol Vol. 147, pgs. 2657-2662 1991) or Fc(RII
(Lund et al., 1991; Sarmay et al., Mol. Immunol., Vol. 29, pgs.
633-639 1992) whereas Gly.sup.238 to Ala has the biggest effect on
binding to Fc(RIII (Sarmay et al., 1992).
[0171] The following samples of DNA were used.
[0172] 1. Plasmid 2555 and Plasmid 2555 Mut.; the humanized V.sub.H
region of NSM 4.7.2.4 cloned into pEE6 expression vector at a Hind
III/Spe I restriction site. Plasmid 2555 then was mutated by site
directed mutagenesis such that amino acid residue Asn.sup.101 is
changed to Asp.sup.101, as shown in SEQ ID Nos.:5 and 6, and SEQ ID
Nos.:21 and 22. The resulting plasmid was plasmid 2555 Mut.
[0173] 2. Plasmid 2798; the humanized V.sub.H region of NSM 4.7.2.4
was joined to human kappa constant regions to give approx 700 bp
fragment cloned into pEE12 expression vector at a Hind III/EcoR
I.
[0174] 3. Plasmid MF4260; the human IgG1 heavy chain was associated
with the humanized CD 18 V.sub.H region, having the mutations
Leu.sup.236 to Ala and Gly.sup.238 to Ala as well as a Spe I
restriction site introduced into framework region 4, cloned into
pUC18.
[0175] The purpose of the Spe I restriction site is to allow
separation and recombination of different variable regions.
[0176] The CD18 V.sub.H region gene is excised from plasmid MF 4260
by digestion with Spe I and Hind III and the remaining vector, now
having only the relevant heavy chain constant region, was purified
using Geneclean. It is ligated with the humanized V.sub.H region
DNA of NSM 4.7.2.4 which has been isolated from plasmid 2555 Mut in
the same way. The product is used to transform "Sure" cells and
colonies are checked for the presence of the expected 1400 bp
complete heavy chain insert.
[0177] The complete V.sub.H and constant region insert was excised
from the pUC vector by digestion with Hind III and EcoR I. The 1400
bp fragment is purified using QiaexII (Qiagen) and then ligated in
turn into the vector pEE6, which has previously been cut with the
same enzymes.
[0178] The next step was to excise the CD4 heavy chain genes from
the pEE6 vector and clone them into pEE12, already containing the
humanized CD4 light chain gene (plasmid 2798). The pEE6 vector was
digested with Sal I and BgI II and the pEE12 vector is digested
with Sal I and BamH I to create the appropriate sites for
re-ligation.
[0179] The final vector construct was checked by restriction
digests with Hind III, EcoR I, Sal I and Spe I for the presence of
the expected fragment, i.e., 700 bp light chain, 1400 bp heavy
chain, 2300 bp fragment of pEE6, and 7000 bp fragment of pEE12.
[0180] The pEE12 vector was linearized by digestion with Sal I and
transfected into NSO cells by electroporation as above. The
transfection mixes were cultured in six 96-well plates, and of 90
growing wells which were tested, all were positive for production
of human heavy and light chains. At this stage a sample of the
pEE12 vector DNA was digested with Sal I and precipitated with
ethanol.
Transfection and Selection of Final Transfectant
[0181] The TRX1 expression vector DNA `pTX/C4` was transfected into
exponentially growing CHO/dhfr- cells that were expanded from the
`Parental CHO DHFR-MCB1`.
[0182] The TRX1 DNA was linearized, and (10) .mu.g of the
linearized TRX1 DNA was added to 1 ml (3.times.10.sup.6 cells) of
exponentially growing CHO/dhfr- cells in an electroporation cuvette
on ice. The cells were transfected (using a BioRad Gene Pulser II)
set to 1000 volts, capacitance of 25 microfarads, and resistance of
.infin. ohms. After electroporation, the cells were placed on ice
for 10 minutes followed by addition to a T25 tissue culture flask
and incubation in a 37.degree. C., 5% CO.sub.2 incubator.
Transformants were selected for the phenotype of neomycin
resistance followed by selection and amplification of DHFR+
transformants using .alpha.-MEM supplemented with methotrexate, 10%
US sourced (harvested in 2001), irradiated, dialyzed FBS, and
neomycin. Cells that survived in the culture medium containing
methotrexate and neomycin were screened for productivity and cloned
by limiting dilution in 96-well plates. These clones were then
screened for high producers. Subclone `E9/3A2` was found to have
the highest specific productivity. This clone was selected and
subsequently expanded for preparation of a pre-seed stock.
[0183] Purification of the Antibody
[0184] Culture supernatant is purified by using a Biopilot
chromatography system (Pharmacia) in three steps as follows: [0185]
(1) Affinity chromatography on a column of Protein A-Sepharose Fast
Flow [0186] (2) Ion exchange chromatography on S-Sepharose Fast
Flow [0187] (3) Size exclusion chromatography on Superdex 20.
[0188] The purified product was filtered and pooled into a single
biocontainer.
[0189] Throughout the purification process, precautions are taken
to ensure that the system remains aseptic. All buffers and reagents
are passed through a 0.2 micron membrane filter and the purified
product is also passed through a 0.2 micron filter before being
pooled. After a batch of antibody has been processed, the entire
chromatography system and columns are sanitized with 0.5M NaOH,
washed with sterile PBS and stored in 20% ethanol. Before it is
used again, the ethanol is washed out with sterile PBS and a
complete trial run is carried out. Samples of buffers and column
eluates are checked for endotoxin level.
Example 2
Construction of TRX1 Antibody Starting from Nucleotide Sequence
[0190] Cloning of Human Constant Regions
[0191] Heavy Chain Constant Region
[0192] The human gamma 1 heavy chain constant region (IgG1) is
amplified from human leukocyte cDNA (QUICK-Clone.TM. cDNA Cat. No.
7182-1, Clontech) using the following primer set and cloned into
pCR-Script (Stratagene). The plasmid containing the human gamma 1
heavy chain constant region in pCR-Script is designated
pHC.gamma.-1. TABLE-US-00008 primer hc.gamma.-1 (SEQ ID No.51) Spe
I 5' primer: 5'- ACT AGT CAC AGT CTC CTC AGC primer hc.gamma.-2
(SEQ ID No.52) EcoR I 3' primer: 5'- GAA TTC ATT TAC CCG GAG ACA
G
[0193] Non-Fc binding mutations (Leu.sup.236 Ala, Gly.sup.38 Ala)
are made in the heavy chain constant region by site-directed
mutagenesis using the following primer and the Transformer.TM.
Site-Directed Mutagenesis Kit from Clontech (Cat. No. K1600-1). The
plasmid containing the human gamma 1 heavy chain non-Fc binding
mutant constant region in pCR-Script is designated
pHC.gamma.-1Fcmut. TABLE-US-00009 primer hc.gamma.-3 (SEQ ID No.53)
Fc mut oligo: 5'- CCG TGC CCA GCA CCT GAA CTC GCG GGG GCA CCG TCA
GTC TTC CTC CCC C
[0194] Light Chain Constant Region
[0195] The human kappa light chain constant region is amplified
from human leukocyte cDNA (QUICK-Clone.TM. cDNA Cat. No. 7182-1,
Clontech) using the following primer set and cloned into pCR-Script
(Stratagene). The plasmid containing the human kappa light chain
constant region in pCR-Script is designated pLCK-1. TABLE-US-00010
primer lc.kappa.-1 (SEQ ID No.54) Kpn I 5' primer: 5'- GGT ACC AAG
GTG GAA ATC AAA CGA AC primer lc.kappa.-2 (SEQ ID No.55) Hind III
3' primer: 5'- AAG CTT CTA ACA CTC TCC CCT GTT G
[0196] Synthesis, Construction and Cloning of TRX1 Variable
Regions
[0197] The heavy and light chain variable regions are constructed
from a set of partially overlapping and complementary synthetic
oligonucleotides encompassing the entire variable regions. The
oligonucleotide set used for each variable region is shown
below.
[0198] Heavy Chain Variable Region Synthetic Oligonucleotides
[0199] Coding Strand Heavy Chain Variable Region Primers
TABLE-US-00011 primer hv-1 (1 - 72) + 6 nucleotide linker (SEQ ID
No.56) 5'- aagctt ATG GAA TGG ATC TGG ATC TTT CTC CTC ATC CTG TCA
GGA ACT CGA GGT GTC CAG TCC CAG GTT CAG CTG GTG primer hv-2 (120 -
193) (SEQ ID No.57) 5'- C TGT AAG GCT TCT GGA TAC ACA TTC ACT GCC
TAT GTT ATA AGC TGG GTG AGG CAG GCA CCT GGA CAG GGC CTT G primer
hv-3 (223 - 292) (SEQ ID No.58) 5'- GGT AGT AGT TAT TAT AAT GAG AAG
TTC AAG GGC AGG GTC ACA ATG ACT AGA GAC ACA TCC ACC AGC ACA G
primer hv-4 (322 - 399) (SEQ ID No.59) 5'- GAG GAC ACT GCG GTC TAT
TAC TGT GCA AGA TCC GGG GAC GGC AGT CGG TTT GTT TAC TGG GGC CAA GGG
ACA CTA GT Non-Coding Strand Heaiy Chain Variable Region Primers
primer hv-S (140 - 51) (SEQ ID No.60) 5'- GTG TAT CCA GAA GCC TTA
CAG GAC ACC TTC ACT GAA GCC CCA GCC TTC TTC ACT TCA GCT CCA GAC TGC
ACC AGC TGA ACC TGG GAC TGG primer hv-6 (246 - 170) (SEQ ID No.61)
5'- CTT CTC ATT ATA ATA ACT ACT ACC GCT TCC AGG ATA AAT CTC TCC CAT
CCA CTC AAG GCC CTG TCC AGG TGC CTG CC primer hv-7 (342 - 272) (SEQ
ID No.62) 5'- GTA ATA GAG CGC AGT GTC CTC AGA CCT GAG GCT GCT GAG
TTG CAT GTA GAC TGT GCT GGT GGA TGT GTC TC
[0200] Light Chain Variable Region Synthetic Oligonucleotides
[0201] Coding Strand Light Chain Variable Region Primers
TABLE-US-00012 primer lv-1 (1 - 63) + 6 nucleotide linker (SEQ ID
No.63) 5'- gaattc ATG GAG ACA GAC ACA ATC CTG CTA TGG GTG CTG CTG
CTC TGG GTT CCA GGC TCC ACT GGT GAC primer lv-2 (93 - 158) (SEQ ID
No.64) 5'- GGC TGT GTC TCT AGG TGA GAG GGC CAC CAT CAA CTG CAA GGC
CAG CCA AAG TGT TGA TTA TGA TGG primer lv-3 (184 - 248) (SEQ ID
No.65) 5'- CAG AAA CCA GGA CAG CCA CCC AAA CTC CTC ATC TAT GTT GCA
TCC AAT CTA GAG TCT GGG GTC CC primer lv-4 (275 - 340) (SEQ ID
No.66) 5'- GGA CAG ACT TCA CCC TCA CCA TCA GTT CTC TGC AGG CGG AGG
ATG TTG CAG TCT ATT ACT GTC AGC Non-Coding Strand Light Chain
Variable Region Primers primer lv-5 (109 - 43) (SEQ ID No.67) 5'-
CAC CTA GAG ACA CAG CCA AAG AAT CTG GAG ATT GGG TCA TCA CAA TGT CAC
CAG TGG AGC CTG GAA C primer lv-6 (203 - 138) (SEQ ID No.68) 5'-
GGT GGC TGT CCT GGT TTC TGT TGA TAC CAG TTC ATA TAA CTA TCA CCA TCA
TAA TCA ACA CTT TGG primer lv-7 (294 - 228) (SEQ ID No.69) 5'- GGT
GAG GGT GAA GTC TGT CCC AGA CCC ACT GCC ACT AAA CCT GTC TGG GAC CCC
AGA CTC TAG ATT G primer lv-8 (378 - 319) (SEQ ID No.70) 5'- GGT
ACC TCC ACC GAA CGT CGG AGG GTC CTG AAG ACT TTG CTG ACA GTA ATA GAC
TGC AAC
[0202] After HPLC purification and removal of organic solvents the
oligonucleotides are resuspended in TE pH8.0 and phosphorylated. An
aliquot of each oligonucleotide in the respective variable region
set then are combined in equal molar amounts. The oligonucleotide
mixtures are heated to 68.degree. C. for 10 minutes and allowed to
cool slowly to room temperature. The annealed oligonucleotides then
are extended to produce double stranded variable region DNA
fragments. For the extension, dNTPs are added to a final
concentration of 0.25 mM followed by an appropriate volume of
5.times.T4 DNA polymerase buffer [165 mM Tris acetate, pH 7.9, 330
mM sodium acetate, 50 mM magnesium acetate, 500 (g/ml BSA, 2.5 mM
DTT] and 4 units of T4 DNA polymerase. The mixture is incubated at
37.degree. C. for 1 hour followed by heat inactivation of the T4
DNA polymerase at 65.degree. C. for 5 minutes.
[0203] The double stranded DNA is ethanol precipitated and
resuspended in the same volume of TE pH 8.0. An appropriate volume
of 5.times.T4 DNA ligase buffer [250 mM Tris-HCl, pH7.6, 50 mM
MgCl.sub.2, 5 mM ATP, 5 mM DTT, 25% w/v polyethylene glycol-8000]
then is added to the double stranded DNA followed by 2 units of T4
DNA ligase and the mixture incubated for 1 hour at 37.degree. C. to
ligate the extended fragments. The T4 DNA ligase then is heat
inactivated at 65.degree. C. for 10 minutes. The variable region
DNA fragments then are phenol extracted, ethanol precipitated, and
resuspended in TE, pH 8.0 and cloned into pCR-Script (Stratagene).
The resulting plasmid containing the heavy chain variable region is
designated pHv-1 and the plasmid containing the light chain
variable region was designated pLV-1.
[0204] The final heavy and light chain expression vectors are
constructed in pcDNA 3.1 (Invitrogen). For the heavy chain
expression vector, the Fc mutated constant region is released from
plasmid pHC-1Fcmut by digestion with Spe I and EcoR I and isolated
by agarose gel electrophoresis. The heavy chain variable region is
released from plasmid pHV-1 by digestion with Hind III and Spe I
and isolated by agarose gel electrophoresis. The two fragments in
equal molar amounts are ligated into the Hind III/EcoR I sites of
pcDNA3.1(+) (Invitrogen) using standard molecular biology
techniques. The resulting TRX1 heavy chain expression vector is
designated pTRX1/HC.
[0205] Similarly, for the light chain expression vector, the light
chain constant region is released from plasmid pLC-1 by digestion
with Kpn I and Hind III followed by agarose gel purification. The
light chain variable region is released from pLV-1 by digestion
with EcoR I and Kpn I followed by agarose gel purification. The two
light chain fragments in equal molar amounts are ligated into the
EcoR I/Hind III sites of pcDNA3.1(-) (Invitrogen) using standard
molecular biology techniques yielding the TRX1 light chain
expression vector pTRX1/LC.
[0206] For production of TRX1 antibody, the TRX1 heavy chain and
TRX1 light chain expression plasmids are cotransfected into CHO
cells using standard molecular biology techniques.
Example 3
Construction of Aglycosylated TRX1 Antibody
[0207] A humanized antibody, e.g., the components of the humanized
antibody, e.g., light chain and heavy chain, each containing
constant regions and variable regions, e.g., amino acid sequences
are shown in Seq ID Nos.: 9, 11, 12, 13, 15, and 16 (FIGS. 2A, 2C,
2D, 2E, and 2G), and were produced by a procedure similar to that
of Example 1. The humanized antibody is an aglycosylated
antibody.
Example 4
Construction of Aglycosylated TRX1 Antibody
[0208] A humanized antibody, e.g., the components of the humanized
antibody, e.g., light chain and heavy chain, each containing
constant regions and variable regions, e.g., amino acid sequences
are shown in Seq ID Nos.: 25, 27, 28, 29, 31, and 32 (FIGS. 4A, 4C,
4D, 4E, and 4G), and is produced by a procedure similar to that of
Example 1. The humanized antibody is an aglycosylated antibody.
Example 5
Treatment of a Primate with TRX1 Antibody
[0209] A baboon having a weight of 4.6 kg received a mismatched
kidney transplant from another baboon on day 1 and was treated with
both the CD4 antibody, e.g., the humanized antibody, e.g., the
components of the humanized antibody, e.g., light chain and heavy
chain, each containing a constant region and a variable region,
e.g., amino acid sequences shown in Seq ID Nos.: 9, 11, 12, 13, 15,
and 16, and with a depleting humanized CD8 antibody, the amino acid
sequences of which is shown in SEQ ID Nos.:33 (FIGS. 5A-5C) and 34
(FIG. 6) (nucleic acid sequences are set forth as SEQ ID Nos.:75
(FIGS. 5A-5C) and 76 (FIG. 6) in accordance with the following
Protocol of Table 1.
[0210] The animal has survived for more than 80 days without
receiving an immunosuppressant. In addition except for a period of
about two days, creatinine levels were below 2 mg/dL.
TABLE-US-00013 TABLE 1 Protocol Study 2 DAYS ACTION 0 1 2 3 4 5 6 7
8 9 10 11 12 13 14 15 Treatments Renal transplantation X CD4
antibody (iv).sup.2 X X X X X X CD8 antibody (iv).sup.3 X X X X X X
.sup.2CD4 antibody 40 mg/kg on day 0 and 20 mg/kg for all other
doses was given by iv infusion over 1 hour .sup.3CD8 antibody 6
mg/kg given as an iv bolus after the CD4 antibody infusion
[0211] These materials and methods were used in the following
examples:
Equine Immunoglobulin as a Source of Antigen.
[0212] Antivenin (Crotalidae polyvalent) was purchased from Fort
Dodge Laboratories (Overland Park, Kans.) and reconstituted with
diluent provided by the manufacturer and used as our source of
equine Ig. The solution was passed through a 2 micron syringe
filter and aggregated by diluting to 25 mg/ml in 0.9% saline and
incubating at 64.degree. C. for 35 min followed by overnight
incubation on ice. The material was stored at -80.degree. C. until
use. The amount of aggregated material in each lot was determined
by HPLC size exclusion chromatography and ranged from 21.2% to
29.9% of total protein.
TRX1 Production and Purification
[0213] TRX1 is derived from the mouse anti-human CD4 hybridoma, NSM
4.7.2.4. The parental heavy and light chain cDNA were cloned from
an NSM 4.7.2.4 cDNA library by cross hybridization with rat heavy
and light chain gene cDNA probes using standard molecular biology
techniques. Sequence analysis of the cDNA derived from NSM 4.7.2.4
confirmed the heavy chain isotype to be gamma-1 and the light chain
kappa. The NSM 4.7.2.4 mouse VH and VL regions were reshaped to
human VH and VL regions using "best fit" or human frameworks with
the highest sequence similarity to that of the mouse VH and VL. For
the light chain, human antibody HSIGKAW (from EMBL) with a sequence
similarity of 79% was used as the target sequence. For the heavy
chain, human antibody A32483 (GenBank) with a sequence similarity
of 74% was used. The humanization was performed by site-directed
mutagenesis of the mouse cDNA clones. To eliminate antibody binding
to Fc receptors as well as complement fixation, a single amino acid
substitution was introduced in the Fc region at amino acid position
297 of .gamma.1 heavy chain constant region by site-directed
mutagenesis eliminating the site of N-linked glycosylation.
[0214] TRX1 antibody was produced at the Therapeutic Antibody
Centre (Oxford, UK) by hollow fiber fermentation of CHO cell
transfectants. The antibody was purified from culture supernatant
by Protein A affinity chromatography followed by cation/anion
exchange, nanofiltration, and finally size exclusion
chromatography. The purified material was formulated in PBS and
stored at -80.degree. C.
Tolerance Induction and Challenge Protocol
[0215] All baboon work was performed at the Southwest Foundation
for Biomedical Research (San Antonio, Tex.) under a protocol
approved by the Institutional Animal Care and Use Committee. Seven
to twenty-one days prior to study, animals were screened by
physical examination, CBC and serum chemistries. Lymphocyte subset
numbers and CD4 expression level on CD3.sup.+ cells were determined
for baseline values. A second set of baseline values was collected
on day -1 just prior to the first TRX1 or saline infusion. Animals
were sedated with a single dose of 10 mg/kg ketamine plus 5 mg
diazepam as needed to facilitate handling. TRX1 and saline
infusions were administered i.v. at a rate of 30 ml/hr.
Temperature, blood pressure and respiration were monitored during
and after infusions. Animals were examined for skin rashes and
lymphadenopathy at the time of each infusion and at the time of
subsequent serum sample collections. In addition animals were
monitored daily for signs of discomfort, malaise, arthralgia and
gastrointestinal complications. The first dose of antigen (equine
Ig) was given on Day 0 as a 10 mg/kg i.v. bolus. All subsequent
doses of equine Ig (Days 4, 8, 68, 95 and 130) were given as a 10
mg/kg s.c. bolus, except for the last challenge on Day 130, which
was a 1 mg/kg s.c. bolus.
[0216] Animals were immunized with SRBC (HemoStat Laboratories,
Dixon, Calif.) to demonstrate immunocompetence to a neo-antigen
after TRX1 exposure. All animals received a single i.v. injection
of a 10% SRBC solution in 0.9% sterile saline at a dose of 1.7
ml/kg on Day 68 of the study.
TRX1 Serum Concentration
[0217] The concentration of TRX1 in serum was determined by ELISA.
50 .mu.l of a 5 .mu.g/ml solution of soluble CD4 in PBS (kindly
provided by the Therapeutic Antibody Centre, Oxford, UK) was
dispensed into 96-well plates and incubated overnight at
2-8.degree. C. After three washes with PBS containing 0.05% Tween
20 (Wash Buffer) plates were blocked with 1% BSA, 0.05% Tween 20 in
PBS (Blocking Buffer) for 1 hr at 37.degree. C. and stored at
2-8.degree. C. Immediately prior to use plates were washed three
times with Wash Buffer. Baboon serum samples were prepared from a
1:10 or 1:100 starting dilution in Blocking Buffer followed by
serial 1:10 dilutions and transfer of 50 .mu.l of diluted sample to
the soluble CD4 coated plates. A standard curve included on each
plate was prepared from a 1 .mu.g/ml solution of unconjugated TRX1
serially diluted 1:4. Following a 2 hr incubation at 37.degree. C.,
plates were washed three times and 50 .mu.l of a
peroxidase-conjugated donkey anti-human IgG (0.08 .mu.g/ml in
Blocking Buffer) was added to each well. Plates were incubated for
1 hr at room temperature, washed three times and developed. TRX1
serum concentrations were calculated from all OD values falling
within the linear portion of the TRX1 standard curve.
Immune Response to Equine Ig
[0218] Baboon anti-globulin responses to equine Ig were determined
by ELISA. 96-well plates coated with 50 .mu.l/well of a 10 .mu.g/ml
solution of antivenin in carbonate buffer were incubated overnight
at 4.degree. C. Plates were then washed three times and blocked for
2 hr at 37.degree. C. Following the blocking step, plates were
washed three times and baboon serum samples added to wells (50
.mu.l/well) using a 3-fold serial dilution scheme beginning with a
1:10 dilution and incubated for 2 hr at room temperature.
[0219] After three washes peroxidase conjugated rabbit anti-human
IgG/IgM antibody (diluted 1/10,000) was added to each well (50
.mu.l/well) and incubated for 1 hr at room temperature. Plates were
then washed three times and 100 .mu.l of substrate added to each
well followed by incubation at room temperature for 8 min. The
assay was standardized by including on each plate a positive
control serum. The positive control serum was obtained from a
previously immunized animal and was used as a standard in all
assays at a dilution of 1:25,000. Titer is defined as the
reciprocal of the dilution resulting in an OD value equivalent to
twice the OD value of a 1:25,000 dilution of the standard.
SRBC Hemolysis Assay
[0220] Immune response to SRBC was assessed by hemolysis. Serum
samples were heat inactivated at 56.degree. C. for 30 min followed
by preparation of a 2-fold dilution series starting from a 1:10
dilution in PBS plus 0.1% BSA. 100 .mu.l of the diluted serum was
combined with an equal volume of a 1% SRBC solution followed by the
addition of 100 .mu.l Guinea pig complement (Sigma-Aldrich)
pre-adsorbed with SRBC diluted 1:10 in PBS. The plates were
incubated at 37.degree. C. for 30 min. Titer is defined as the
reciprocal of the highest dilution of serum that did not cause
obvious hemolysis.
Antibodies and Flow Cytometry
[0221] Normal donkey serum, donkey anti-human IgG-biotin, donkey
anti-human IgG F(ab').sub.2-biotin, donkey anti-human
IgG-peroxidase, donkey IgG-biotin, rabbit anti-human IgG/IgM and
human IgG-biotin were purchased from Jackson ImmunoResearch. FITC
conjugated mouse anti-human CD4, clone M-T441, and FITC conjugated
mouse IgG2b, clone BPC 4, were purchased from Ancell, Inc. Mouse
anti-human CD3 FITC, clone SP34, mouse IgG3 FITC, and mouse
anti-human CD45RA-PE were purchased from BD Pharmingen. Mouse
anti-human CD8-PerCP and mouse IgG1-PerCP were purchased from BD
Biosciences. Streptavidin-Quantum Red was purchased from
Sigma-Aldrich and FITC and Cy5 conjugated standard beads from Bangs
Laboratories (Fishers, Ind.).
[0222] CD4 saturation was determined as a function of free CD4
sites on circulating lymphocytes. 100 .mu.l of heparinized whole
blood was pelleted by centrifugation and plasma removed by
aspiration. Cells were re-suspended in 100 .mu.l of a 1.0 .mu.g/ml
solution of biotinylated TRX1 or biotinylated human IgG. Following
a 20 min incubation on ice cells were washed with 1 ml of Wash
Buffer and incubated with 50 .mu.l Streptavidin Quantum Red (1:5
dilution of stock) for 20 min on ice. RBC were then lysed by the
addition of 2 ml of Lysis Buffer (0.15M NH.sub.4Cl, 10 mM
KHCO.sub.3, 100 .mu.M disodium EDTA). Samples were vortexed and
incubated at room temperature until clear (approximately 10 min).
RBC debris was removed by centrifugation and washing with 1 ml Wash
Buffer. Cells were fixed by the addition of PBS, 0.1% formalin.
Intra-day fluorescence sensitivity variation was controlled by
using FITC and Cy5 conjugated standard beads.
CD4.sup.+ lymphocyte counts
[0223] The number of CD4.sup.+ lymphocytes in peripheral blood was
determined by multiplying the absolute lymphocyte count obtained
from CBC data by the percentage of CD4.sup.+ lymphocytes. The
percentage of CD4.sup.+ lymphocytes in whole blood was determined
by flow cytometry as the percentage of CD4.sup.+ cells in the
lymphocyte gate staining with FITC-conjugated M-T441, a mouse
antibody recognizing domain 2 of CD4 that does not compete with
TRX1 binding to CD4. The TRX1 antibody used in these examples was a
humanized IgG1 antibody recognizing domain 1 of human CD4 further
modified by introducing a single amino acid substitution (Asn to
Ala) at position 297 in the heavy chain constant region, so
eliminating a major glycoslyation site necessary for high affinity
Fc receptor interactions and complement binding (Bolt, S., E. et
al. 1993. Eur. J. Immunol. 23:403; Friend, P. J., et al. 1997.
Transplantation 68:1632; Routledge, E. G., et al. 1995.
Transplantation 60:847).
Study Design
[0224] To identify a model species in which to test tolerance
induction with TRX1, a number of non-human primates were screened
including African green monkey, cynomolgus and rhesus macaque,
baboon and chimpanzee, for cross-reactivity with TRX1. While all
showed some degree of immunoreactivity, only in chimpanzee and
baboon was the binding affinity comparable to human. Thus, baboon
was selected as the model species.
[0225] As a target antigen for tolerance induction, a simple, yet
clinically relevant, model antigen was sought. This allowed testing
for antigen specific tolerance as well as optimization of the
induction protocol prior to investigation in more complex models
such as organ transplant and autoimmune diseases. A well
characterized immunogenic biologic antivenin, or anti-venom,
Crotalidae polyvalent was selected--a commercial preparation of
equine immune serum globulins (equine Ig) isolated from horses
immunized with pit viper venoms (Jurkovich, G. J., et al., 1988. J
Trauma 28:1032; Dart, R. C., and J. McNally. 2001. Ann. Emerg. Med.
37:181). To ensure immunogenicity of the equine Ig the material was
heat-aggregated and the preparation tested in a pilot experiment to
determine a dose and route of administration that would generate a
robust immune response prior to use in the tolerance induction
protocol.
[0226] To investigate the feasibility of tolerance induction with
TRX1 in baboons an experimental protocol divided into 3
phases--induction, washout, and challenge (FIG. 9A) was designed
and implemented by assigning twenty-one baboons (Papio cynocephalus
anubis) to one of 7 groups (3 animals/group) including 4
experimental and 3 control groups (FIG. 9B). The experimental arm
of the induction phase consisted of 4 TRX1 dosing cohorts of 1, 10,
20, or 40 mg/kg per dose infused 4 times over 13 days on day -1,
day 3 or 4, day 8 and day 12. A 10 mg/kg i.v. bolus of heat
aggregated antigen (equine Ig) was delivered on day 0 followed on
days 4 and 8 with a s.c. bolus of the same dose. In the control
arm, animals in control group I (antigen only), were infused with
an equivalent volume of normal saline instead of TRX1 at each time
point exactly as animals in the experimental groups. Control group
II, (TRX1 only), was comprised of 2 cohorts, 20 mg/kg and 40 mg/kg
TRX1, dosed on the same schedule as the experimental groups but
receiving normal saline instead of equine Ig during the
tolerization phase. TRX1 serum concentrations were determined 24
hours after the first dose of antibody and immediately prior to the
3 subsequent doses as well as weekly thereafter. Serum levels of
TRX1 and equine Ig were monitored until no longer detectable
(washout phase), at which time all animals were challenged by s.c.
injection with heat-aggregated equine Ig (challenge phase).
Example 6
TRX1 Suppresses the Humoral Response During Induction without
Depletion of T-Cells
[0227] A dose dependent increase in TRX1 serum concentration was
evident 24 hours after the first dose ranging from a mean of
15.6.+-.4.1 .mu.g/ml (n=3) in animals receiving 1 mg/kg up to a
mean of 542.5.+-.138.1 .mu.g/ml (n=6) in those receiving 40 mg/kg
(FIG. 10A). Serum concentrations of TRX1 determined immediately
prior to subsequent doses indicated a dose accumulation of TRX1 in
the 20 mg/kg and 40 mg/kg treated animals with mean trough level
concentrations increasing after each dose. Minimum TRX1 serum
concentrations occurred between the first and second dose of
antibody and ranged from a mean of 39.4.+-.18.0 .mu.g/ml (n=6) for
20 mg/kg TRX1 treated animals up to a mean of 162.+-.63.3 .mu.g/ml
(n=6) for those receiving 40 mg/kg of TRX1. There was no dose
accumulation of TRX1 in animals receiving 1 mg/kg or 10 mg/kg TRX1
as trough level concentrations determined immediately prior to the
last three doses of antibody were below the limit of detection of
the assay (0.2 ng/ml) as were those in control group I animals,
i.e., those receiving antigen only. A protocol deviation at the
time of the second TRX1 infusion eliminated one animal (#16250)
from further study in the 20 mg/kg TRX1 only control group II.
[0228] TRX1 was detected by flow cytometry on CD3.sup.+ lymphocytes
using biotinylated F(ab').sub.2 donkey anti-human IgG. Twenty four
hours after the first infusion MCF values were well above baseline
values and remained so throughout the treatment period beginning a
return to baseline levels at day 27. TRX1 was undetectable on cells
by day 48. To determine the level of CD4 saturation by TRX1,
biotinylated TRX1 was added to whole blood samples and cell
staining assessed by flow cytometry (FIG. 10B). As expected from
the TRX1 serum concentration data, free CD4 sites were readily
detected in the 1 mg/kg TRX1 group. Except for the initial 24-hr
time point, MCF values determined for samples obtained just prior
to TRX1 dosing on days 3, 8 and 12 in the 1 mg/kg group, were only
slightly below baseline values averaging 89.5% of baseline (range,
86.0%-92.9%), or 10.5% saturated. Free binding sites were also
detected in the 10 mg/kg TRX1 group from samples taken just before
TRX1 dosing on days 3, 8 and 12 with an average MCF value of 25.8%
of baseline (range, 19.3%-33.4%) during the induction phase,
indicating 74.2% of the sites were saturated. The 20 mg/kg group
averaged 14.9% of baseline MCF staining (range, 10.2%-18.2%), or
85.1% saturated, during the induction phase whereas the 40 mg/kg
group averaged MCF values of 9.5% of baseline (range 8.1%-10.7%),
or 90.5% saturated. By day 20, one week after the last dose of
TRX1, MCF values for the both 1 mg/kg and 10 mg/kg TRX1 groups had
returned to baseline, while staining from the 20 mg/kg TRX1 group
indicated the number of free CD4 sites at approximately 25% of
baseline. The 40 mg/kg TRX1 group maintained maximum saturation at
day 20, but free CD4 sites were detected on day 27 with average MCF
values at 24.7% of baseline, reflecting 75.3% saturation. By day 48
MCF values had returned to baseline for both the 20 mg/kg and 40
mg/kg TRX1 groups. Reappearance of free CD4 sites correlated with
the reduction in TRX1 serum concentrations during the washout phase
with biotinylated TRX1 staining first beginning to increase once
TRX1 serum levels dropped below approximately 10 .mu.g/ml.
[0229] One animal in the 20 mg/kg TRX1 experimental group (#15983)
showed a more rapid return to baseline of free CD4 sites as well as
a more rapid clearance of TRX1 from the serum. That this was due to
the development of an immune response against TRX1 was subsequently
confirmed by ELISA. Of note this animal had the lowest TRX1 serum
concentration trough level of all animals in the 20 mg/kg TRX1
group, 13.4 .mu.g/ml on day 4, between the first and second dose of
antibody. All other animals in the group had TRX1 serum
concentrations.gtoreq.35.0 .mu.g/ml. Data from this animal are not
included in the 20 mg/kg group mean calculations. All animals in
the 1 mg/kg (3/3) and 10 mg/kg (3/3) TRX1 experimental groups made
an immune response to TRX1 detectable by ELISA 7-10 days after the
first dose of antibody. Only one other animal (#16313) made a
detectable immune response to TRX1, this occurring in the 40 mg/kg
TRX1 control group II. However, this response was not detectable
until day 27, more than 2 weeks after the last dose of TRX1.
[0230] No treatment-related adverse events were observed during
infusions or at any time after TRX1 dosing for the duration of the
study. Complete blood counts and flow cytometry data showed no
apparent depletion of CD4.sup.+ lymphocytes at any dose. While
day-to-day variability in lymphocyte counts were evident, no
significant differences between TRX1 treated animals and those
receiving saline were observed, nor were any dose dependent
differences evident among the TRX1 treated animals (FIG. 10C).
Similar to the in vitro assessment, only modest CD4 modulation from
the cell surface was observed.
[0231] Administration of TRX1 did result in a dose dependent
inhibition of the humoral response to equine Ig during the
induction and washout phases (FIG. 11A). No immune response to
equine Ig was detected in any animals in the 40 mg/kg TRX1
experimental group throughout this period. However, an elevation in
the group mean titers against equine Ig was evident for the 20
mg/kg TRX1 experimental group. Two of 3 animals in this group,
#16276 and #16096, responded with maximum peak titers of less than
10-fold above baseline, this occurring on day 27 followed by a
return to baseline by day 48. Animal #15983, the same animal in
which an immune response to TRX1 was observed, mounted a larger and
more sustained response to equine Ig during the induction and
washout phases peaking on day 41 at >25-fold above baseline and
remaining >10-fold above baseline through the washout phase.
Higher titers were also evident in both the 1 mg/kg and 10 mg/kg
TRX1 experimental groups as well as in control group I (antigen
only). Surprisingly, mean titers for the 1 mg/kg TRX1 experimental
group were approximately 10- to 15-fold above those for control
group I. One explanation for this apparently enhanced response may
be priming to human Ig epitopes cross-reactive with equine Ig.
Example 7
TRX1 Induces Antigen Specific Hyporesponsiveness and Tolerance
[0232] Once TRX1 serum levels fell below the limit of detection,
tolerance to equine Ig was assessed by challenging animals with
immunogenic, heat-aggregated antigen and measuring the resulting
specific humoral immune response. Animals were first challenged by
s.c. administration of 10 mg/kg of equine Ig on day 68. All animals
in the 1 mg/kg and 10 mg/kg TRX1 dose groups generated a robust
secondary immune response equine Ig with group mean antibody titers
closely matching that of control group I (FIG. 11B). The response
was characterized by a rapid rise in antibody titer as well as
higher maximum titers compared with the response observed in these
groups during the tolerization phase. Showing no evidence of
tolerance to equine Ig, animals from the 1 mg/kg and 10 mg/kg TRX1
experimental groups were released from study after the first
challenge. Control group II, receiving antigen for the first time
on day 68, responded with a group mean antibody titer to equine Ig
rising more slowly than the recall response in control group I
(FIG. 11B), as would be expected of a primary response. Group mean
titers for the 20 mg/kg the 40 mg/kg TRX1 experimental groups also
increased in response to challenge but with significantly reduced
(50- to 250-fold) peak titers compared to control group I (FIG.
11B). One of three animals in the 20 mg/kg TRX1 experimental group
responded to challenge with a rise in titer similar to control
group I, this occurring in animal #15983, which had also generated
an immune response to TRX1 during the induction and washout phases.
The two other animals in this group, #16276 and #16096, were
hyporesponsive to challenge with a maximum mean peak response
10-fold less than control group I. In the 40 mg/kg TRX1
experimental group one animal, #16192, was similarly hyporesponsive
to challenge with the two other animals in this group, #16178 and #
16286, showing no response to challenge.
[0233] To demonstrate that the failure to mount a vigorous immune
response upon antigen challenge in the 20 mg/kg and 40 mg/kg TRX1
experimental groups was antigen specific and not the consequence of
treatment related immune suppression, immunocompetence was assessed
by immunizing all animals with a third-party-antigen, SRBC, at the
time of first challenge on day 68. All groups mounted an
essentially equivalent anti-SRBC hemolytic response to this
challenge (FIG. 11C), which was confirmed to be predominately IgG
by ELISA.
[0234] Control groups I and II as well as the 20 mg/kg and 40 mg/kg
TRX1 experimental groups were re-challenged with 10 mg/kg equine Ig
on day 95 and again on day 130 with 1 mg/kg equine Ig (FIG. 12A).
All control groups, antigen only and TRX1 only, showed a similar
boost in the humoral response to equine Ig further demonstrating
that TRX1 treatment alone did not induce long-standing immune
suppression. However, group mean titers for the 20 mg/kg and 40
mg/kg experimental groups failed to rise above the maximum peak
titers of the first challenge even with repeated challenges. For
animals in the 20 mg/kg TRX1 experimental group, excluding animal
#15983, maximum titers occurred after the first challenge with peak
titers of 269 and 145 for animals #16096 and #16276, respectively.
Peak responses then diminished upon repeated challenge to 35 and
92, respectively, after the third challenge (FIG. 12A). Group mean
titers in the 40 mg/kg TRX1 experimental group were consistently
lower than those of the 20 mg/kg group with a single animal,
#16192, accounting for essentially all of the response with a
maximum peak titer of 313 after the first challenge. As with the
animals in 20 mg/kg TRX1 group the peak response to each subsequent
challenge was lower than for the previous challenge with animal
#16192 response declining to a peak titer of only 39 after the
third challenge with antigen (FIG. 12B). The two other animals in
the 40 mg/kg TRX1 experimental group, #16178 and #16286, generated
virtually no detectable immune response to equine Ig upon repeated
challenge (FIG. 12B).
[0235] A second study (3 animals/group) was performed with the 20
mg/kg TRX1 dose reducing the number of TRX1 doses from 4 to 3 but
administering them every other day on days -1, 1 and 3. A control
group (control group I) was also included with animals receiving
saline infusions in place of TRX1. Equine Ig dosing was unchanged
with the animals receiving 3 doses of 10 mg/kg on days 0, 3 and 8.
As in the first study, TRX1 administration resulted in a
suppression of the humoral response to equine Ig during induction
and washout phases compared to control group I with one animal,
#16224, accounting for essentially all of the detectable response
(FIG. 13A). On day 68 with serum levels of TRX1 below detectable
levels animals were challenged with equine Ig Control group I
animals responded as expected with a rapid and robust rise in titer
to a mean peak response of 7652. In the 20 mg/kg TRX1 treated
group, animal #16224 showed a rapid rise in titer similar to
control group animals with a maximum peak titer of 6139. However,
two other animals in the group, #12093 and #16130, were
hyporesponsive to challenge generating peak titers of 37 and 161,
respectively. A second challenge on day 97 produced only a slight
rise in titer to 20 and 26 for animals #12093 and #16130,
respectively, which fell rapidly to baseline. These two animals
showed no response to a third challenge with antigen. As in the
previous study all animals responded to SRBC neoantigen
immunization at the time of first challenge on day 68.
[0236] By increasing the TRX1 dose to 20 mg/kg hyporesponsiveness
was induced in two of three animals with the maximum response titer
diminishing after each subsequent challenge. At doses of 40 mg/kg
two of three animals were completely non-responsivene to multiple
antigen challenges and the third hyporesponsive to antigen with
peak response titers again declining with each antigen-challenge.
Studies in mice have demonstrated that 3 doses of 20-25 mg/kg of a
non-depleting anti-CD4 antibody administered every other day were
sufficient to induce tolerance, although the time required for
tolerance to become evident was approximately 1 month after dose
initiation. The 20 mg/kg TRX1 dosing was modified, administering 3
doses, one every other day, beginning one day before antigen
administration. With this modification two of three animals became
completely unresponsive to antigen challenge after an initial
period of hyporesponsiveness.
[0237] In man reduced immunogenicity and improved pharmacokinetics
may support a lower efficacious dose of TRX1. For example, while an
immune response against TRX1 has been detected in all baboons
receiving only a single dose of the antibody (n=9), no immune
response to TRX1 after a single dose of the antibody has been
detected in man (n=9). Furthermore, a 2.5 fold increase in the
serum half-life of TRX1 in man should allow for sustained CD4
coverage with less antibody compared to baboon.
[0238] No acute adverse events were associated with any dose of
TRX1, and those dosing regimens that resulted in hyporesponsiveness
and tolerance, while clearly immunosuppressive during the induction
phase, were not associated with any clinical or histopathologic
side effects. TRX1 treated animals were not housed in isolation or
in germ free or specific pathogen free conditions. Despite
virtually complete saturation of CD4 sites on peripheral
lymphocytes for at least 21 days, no evidence for increased
prevalence of enteric parasites or opportunistic bacterial, fungal,
or viral infections or recrudescence of endogenous virus was found
during TRX1 treatment or at any time thereafter.
[0239] It is noted that the failure of TRX1 to induce
self-tolerance in the control group II animal #16313 may be due to
acute infection during the tolerance induction phase with SA8
virus, an alphaherpesvirus prevalent in the baboon colony from
which all animals in the study were obtained. Animal #16313 became
seropositve to SA8 during the induction phase, while all other
animals were either seropositive before the study, or remained
seronegative throughout the study.
Example 8
Anti-CD4 Antibody has Effects on Monocytes/Macrophages
[0240] In this example human peripheral blood monocytes were
incubated with nothing, anti-CD4 (TRX1) human IgG or aglycosyl CD8
antibody for 3, 4, or 5 days. RNA was analyzed by qualitative PCR
for the levels of Fc.gamma.RIIa and F.gamma.gRIIB message. TRX1
incubation was found to increase the level of Fc.gamma.RIIa and
Fc.gamma.RIIb message.
[0241] TRX1-treated human monocyte/macrophages were also stained
for CD14, CD83, CD16, CD32, CD80, CD86, MHCII, CD11b, CD62L, CCR2,
and CXCR4. The phenotype of the treated cells was determined to be
CD14 dim with reduced expression of CD86, CD11b, CCR2 and CXCR4.
The cells had increased expression of CC16 (Fc.gamma.RIII), CD32
(Fc.gamma.RII) and MHC class II. The maximum effect on expression
of Fc.gamma.RIIb (which is known to inhibit inflammatory signals
delivered by Fc.gamma.RIIa and Fc.gamma.RIII) was observed after 4
to 7 days.
[0242] CD4 treated murine monocyte/macrophages also made lower
amounts of inflammatory cytokines (e.g., IL4, IFN.gamma., GM-CSF)
and more cytokines associated with development of Treg cells (e.g.,
IL-10 and TGF.beta.).
[0243] Human monocytes were prepared from whole blood by isolating
PBMC on Ficoll and then separating the monocytes from the
lymphocytes using a negative selection kit composed of magnetic
beads coupled with antibodies recognizing all cells types but
monocytes and then removal of the tagged cells on a MACS cell
sorting machine. Purified monocytes were incubated with nothing,
human IgG (100 ug/ml or 50 ug/ml), TRX1 (50 ug/ml or 10 ug/ml) or
aglycosyl anti-human CD8 antibody (50 ug/ml or 10 ug/ml) for 5
days. At that point, the cells were washed 3 times in fresh medium
to remove any residual antibody and plated with allogenic purified
human T cells (also purified using magnetic beads in a negative
selection process) at a ratio of 2 T cells: 1 monocyte. After 5
days the cultures were fed with medium containing .sup.3H-thymidine
to measure dividing cells and cultures were harvested 18 hrs later.
Data were expressed as the percent of thymidine incorporated in
wells containing untreated monocytes and T cells. While anti-CD8
antibody had no effect on the MLR response (or slightly increased
the response) anti-TRX1 at both 50 and 10 ug/ml reduced the
response to levels below 20% of control. The human IgG at 50 ug/ml
response was about 90% of control.
[0244] Numerous modifications and variations of the invention are
possible in light of the above teachings; therefore, within the
scope of the appended claims, the invention may be practiced
otherwise than as particularly described.
Equivalents
[0245] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, numerous
equivalents to the specific polypeptides, nucleic acids, methods,
assays and reagents described herein. Such equivalents are
considered to be within the scope of this invention and are covered
by the following claims.
Sequence CWU 1
1
76 1 238 PRT Artificial Sequence Description of Artificial Sequence
Synthetic TRX1 antibody light chain construct 1 Met Glu Thr Asp Thr
Ile Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr
Gly Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala 20 25 30 Val
Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser 35 40
45 Val Asp Tyr Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro
50 55 60 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Val Ala Ser Asn Leu
Glu Ser 65 70 75 80 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr 85 90 95 Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp
Val Ala Val Tyr Tyr Cys 100 105 110 Gln Gln Ser Leu Gln Asp Pro Pro
Thr Phe Gly Gly Gly Thr Lys Val 115 120 125 Glu Ile Lys Arg Thr Val
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro 130 135 140 Ser Asp Glu Gln
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu 145 150 155 160 Asn
Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn 165 170
175 Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
180 185 190 Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser
Lys Ala 195 200 205 Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val
Thr His Gln Gly 210 215 220 Leu Ser Ser Pro Val Thr Lys Ser Phe Asn
Arg Gly Glu Cys 225 230 235 2 717 DNA Artificial Sequence
Description of Artificial Sequence Synthetic TRX1 antibody light
chain construct 2 atg gag aca gac aca atc ctg cta tgg gtg ctg ctg
ctc tgg gtt cca 48 Met Glu Thr Asp Thr Ile Leu Leu Trp Val Leu Leu
Leu Trp Val Pro 1 5 10 15 ggc tcc act ggt gac att gtg atg acc caa
tct cca gat tct ttg gct 96 Gly Ser Thr Gly Asp Ile Val Met Thr Gln
Ser Pro Asp Ser Leu Ala 20 25 30 gtg tct cta ggt gag agg gcc acc
atc aac tgc aag gcc agc caa agt 144 Val Ser Leu Gly Glu Arg Ala Thr
Ile Asn Cys Lys Ala Ser Gln Ser 35 40 45 gtt gat tat gat ggt gat
agt tat atg aac tgg tat caa cag aaa cca 192 Val Asp Tyr Asp Gly Asp
Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro 50 55 60 gga cag cca ccc
aaa ctc ctc atc tat gtt gca tcc aat cta gag tct 240 Gly Gln Pro Pro
Lys Leu Leu Ile Tyr Val Ala Ser Asn Leu Glu Ser 65 70 75 80 ggg gtc
cca gac agg ttt agt ggc agt ggg tct ggg aca gac ttc acc 288 Gly Val
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 85 90 95
ctc acc atc agt tct ctg cag gcg gag gat gtt gca gtc tat tac tgt 336
Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys 100
105 110 cag caa agt ctt cag gac cct ccg acg ttc ggt gga ggt acc aag
gtg 384 Gln Gln Ser Leu Gln Asp Pro Pro Thr Phe Gly Gly Gly Thr Lys
Val 115 120 125 gaa atc aaa cga act gtg gct gca cca tct gtc ttc atc
ttc ccg cca 432 Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro 130 135 140 tct gat gag cag ttg aaa tct gga act gcc tct
gtt gtg tgc ctg ctg 480 Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser
Val Val Cys Leu Leu 145 150 155 160 aat aac ttc tat ccc aga gag gcc
aaa gta cag tgg aag gtg gat aac 528 Asn Asn Phe Tyr Pro Arg Glu Ala
Lys Val Gln Trp Lys Val Asp Asn 165 170 175 gcc ctc caa tcg ggt aac
tcc cag gag agt gtc aca gag cag gac agc 576 Ala Leu Gln Ser Gly Asn
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser 180 185 190 aag gac agc acc
tac agc ctc agc agc acc ctg acg ctg agc aaa gca 624 Lys Asp Ser Thr
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala 195 200 205 gac tac
gag aaa cac aaa gtc tac gcc tgc gaa gtc acc cat cag ggc 672 Asp Tyr
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly 210 215 220
ctg agc tcg ccc gtc aca aag agc ttc aac agg gga gag tgt tag 717 Leu
Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 3
238 PRT Artificial Sequence Description of Artificial Sequence
Synthetic TRX1 antibody light chain construct 3 Met Glu Thr Asp Thr
Ile Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr
Gly Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala 20 25 30 Val
Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser 35 40
45 Val Asp Tyr Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro
50 55 60 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Val Ala Ser Asn Leu
Glu Ser 65 70 75 80 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr 85 90 95 Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp
Val Ala Val Tyr Tyr Cys 100 105 110 Gln Gln Ser Leu Gln Asp Pro Pro
Thr Phe Gly Gly Gly Thr Lys Val 115 120 125 Glu Ile Lys Arg Thr Val
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro 130 135 140 Ser Asp Glu Gln
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu 145 150 155 160 Asn
Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn 165 170
175 Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
180 185 190 Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser
Lys Ala 195 200 205 Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val
Thr His Gln Gly 210 215 220 Leu Ser Ser Pro Val Thr Lys Ser Phe Asn
Arg Gly Glu Cys 225 230 235 4 218 PRT Artificial Sequence
Description of Artificial Sequence Synthetic TRX1 antibody light
chain construct 4 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala
Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser
Gln Ser Val Asp Tyr Asp 20 25 30 Gly Asp Ser Tyr Met Asn Trp Tyr
Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Val
Ala Ser Asn Leu Glu Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu
Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Ser Leu 85 90 95
Gln Asp Pro Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100
105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn
Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp
Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr
Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr
Lys Ser Phe Asn Arg Gly Glu Cys 210 215 5 467 PRT Artificial
Sequence Description of Artificial Sequence Synthetic TRX1 antibody
heavy chain construct 5 Met Glu Trp Ile Trp Ile Phe Leu Leu Ile Leu
Ser Gly Thr Arg Gly 1 5 10 15 Val Gln Ser Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Ala Tyr Val Ile
Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Met
Gly Glu Ile Tyr Pro Gly Ser Gly Ser Ser Tyr Tyr Asn 65 70 75 80 Glu
Lys Phe Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser 85 90
95 Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
100 105 110 Tyr Tyr Cys Ala Arg Ser Gly Asp Gly Ser Arg Phe Val Tyr
Trp Gly 115 120 125 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser 130 135 140 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala 145 150 155 160 Ala Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val 165 170 175 Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 180 185 190 Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 195 200 205 Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 210 215
220 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
225 230 235 240 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Ala Gly 245 250 255 Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 275 280 285 Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 305 310 315 320 Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 325 330 335
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 340
345 350 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val 355 360 365 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser 370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430 Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445 His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460
Pro Gly Lys 465 6 1404 DNA Artificial Sequence Description of
Artificial Sequence Synthetic TRX1 antibody heavy chain construct 6
atg gaa tgg atc tgg atc ttt ctc ctc atc ctg tca gga act cga ggt 48
Met Glu Trp Ile Trp Ile Phe Leu Leu Ile Leu Ser Gly Thr Arg Gly 1 5
10 15 gtc cag tcc cag gtt cag ctg gtg cag tct gga gct gaa gtg aag
aag 96 Val Gln Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys 20 25 30 cct ggg gct tca gtg aag gtg tcc tgt aag gct tct gga
tac aca ttc 144 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe 35 40 45 act gcc tat gtt ata agc tgg gtg agg cag gca
cct gga cag ggc ctt 192 Thr Ala Tyr Val Ile Ser Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu 50 55 60 gag tgg atg gga gag att tat cct gga
agc ggt agt agt tat tat aat 240 Glu Trp Met Gly Glu Ile Tyr Pro Gly
Ser Gly Ser Ser Tyr Tyr Asn 65 70 75 80 gag aag ttc aag ggc agg gtc
aca atg act aga gac aca tcc acc agc 288 Glu Lys Phe Lys Gly Arg Val
Thr Met Thr Arg Asp Thr Ser Thr Ser 85 90 95 aca gtc tac atg gaa
ctc agc agc ctg agg tct gag gac act gcg gtc 336 Thr Val Tyr Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 tat tac tgt
gca aga tcc ggg gac ggc agt cgg ttt gtt tac tgg ggc 384 Tyr Tyr Cys
Ala Arg Ser Gly Asp Gly Ser Arg Phe Val Tyr Trp Gly 115 120 125 caa
ggg aca cta gtc aca gtc tcc tca gcc tcc acc aag ggc cca tcg 432 Gln
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 130 135
140 gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggg ggc aca gcg
480 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
145 150 155 160 gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccg
gtg acg gtg 528 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val 165 170 175 tcg tgg aac tca ggc gcc ctg acc agc ggc gtg
cac acc ttc ccg gct 576 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala 180 185 190 gtc cta cag tcc tca gga ctc tac tcc
ctc agc agc gtg gtg acc gtg 624 Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val 195 200 205 ccc tcc agc agc ttg ggc acc
cag acc tac atc tgc aac gtg aat cac 672 Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His 210 215 220 aag ccc agc aac acc
aag gtg gac aag aaa gtt gag ccc aaa tct tgt 720 Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 225 230 235 240 gac aaa
act cac aca tgc cca ccg tgc cca gca cct gaa ctc gcg ggg 768 Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Ala Gly 245 250 255
gca ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc atg 816
Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260
265 270 atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc
cac 864 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His 275 280 285 gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc
gtg gag gtg 912 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val 290 295 300 cat aat gcc aag aca aag ccg cgg gag gag cag
tac aac agc acg tac 960 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr 305 310 315 320 cgt gtg gtc agc gtc ctc acc gtc
ctg cac cag gac tgg ctg aat ggc 1008 Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly 325 330 335 aag gag tac aag tgc
aag gtc tcc aac aaa gcc ctc cca gcc ccc atc 1056 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 340 345 350 gag aaa
acc atc tcc aaa gcc aaa ggg cag ccc cga gaa cca cag gtg 1104 Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360
365 tac acc ctg ccc cca tcc cgg gat gag ctg acc aag aac cag gtc agc
1152 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
Ser 370 375 380 ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc
gcc gtg gag 1200 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 385 390 395 400 tgg gag agc aat ggg cag ccg gag aac
aac tac aag acc acg cct ccc 1248 Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 gtg ctg gac tcc gac ggc
tcc ttc ttc ctc tac agc aag ctc acc gtg 1296 Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430 gac aag agc
agg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg atg 1344 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445
cat gag gct ctg cac aac cac tac acg cag aag agc ctc tcc ctg tct
1392 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 450 455 460 ccg ggt aaa tga 1404 Pro Gly Lys 465 7 467 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
TRX1 antibody heavy chain construct 7 Met Glu Trp Ile Trp Ile Phe
Leu Leu Ile Leu Ser Gly Thr Arg Gly 1 5 10 15 Val Gln Ser Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala
Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe 35 40 45 Thr Ala Tyr Val Ile Ser Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Met Gly Glu Ile Tyr Pro
Gly Ser Gly Ser Ser Tyr Tyr Asn 65 70 75 80 Glu Lys Phe Lys Gly Arg
Val Thr Met Thr Arg Asp Thr Ser Thr Ser 85 90 95 Thr Val Tyr Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr
Cys Ala Arg Ser Gly Asp Gly Ser Arg Phe Val Tyr Trp Gly 115 120 125
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 130
135 140 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala 145 150 155 160 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val 165 170 175 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala 180 185 190 Val Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val 195 200 205 Pro Ser Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His 210 215 220 Lys Pro Ser Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 225 230 235 240 Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Ala Gly 245 250
255 Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
260 265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 275 280 285 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 305 310 315 320 Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 370 375
380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro 405 410 415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460 Pro Gly Lys 465 8
448 PRT Artificial Sequence Description of Artificial Sequence
Synthetic TRX1 antibody heavy chain construct 8 Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ala Tyr 20 25 30 Val
Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45 Gly Glu Ile Tyr Pro Gly Ser Gly Ser Ser Tyr Tyr Asn Glu Lys Phe
50 55 60 Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr
Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Asp Gly Ser Arg Phe Val
Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170
175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Ala Gly Ala Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295
300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420
425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 435 440 445 9 238 PRT Artificial Sequence Description of
Artificial Sequence Synthetic TRX1 antibody light chain construct 9
Met Glu Thr Asp Thr Ile Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5
10 15 Gly Ser Thr Gly Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu
Ala 20 25 30 Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ala
Ser Gln Ser 35 40 45 Val Asp Tyr Asp Gly Asp Ser Tyr Met Asn Trp
Tyr Gln Gln Lys Pro 50 55 60 Gly Gln Pro Pro Lys Leu Leu Ile Tyr
Val Ala Ser Asn Leu Glu Ser 65 70 75 80 Gly Val Pro Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr 85 90 95 Leu Thr Ile Ser Ser
Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys 100 105 110 Gln Gln Ser
Leu Gln Asp Pro Pro Thr Phe Gly Gly Gly Thr Lys Val 115 120 125 Glu
Ile Lys Arg Thr Val Ala Ala Leu Ser Val Phe Ile Phe Pro Pro 130 135
140 Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu
145 150 155 160 Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys
Val Asp Asn 165 170 175 Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val
Thr Glu Gln Asp Ser 180 185 190 Lys Asp Ser Thr Tyr Ser Leu Ser Ser
Thr Leu Thr Leu Ser Lys Ala 195 200 205 Asp Tyr Glu Lys His Lys Val
Tyr Ala Cys Glu Val Thr His Gln Gly 210 215 220 Leu Ser Ser Pro Val
Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 10 717 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
TRX1 antibody light chain construct 10 atg gag aca gac aca atc ctg
cta tgg gtg ctg ctg ctc tgg gtt cca 48 Met Glu Thr Asp Thr Ile Leu
Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 ggc tcc act ggt gac
att gtg atg acc caa tct cca gat tct ttg gct 96 Gly Ser Thr Gly Asp
Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala 20 25 30 gtg tct cta
ggt gag agg gcc acc atc aac tgc aag gcc agc caa agt 144 Val Ser Leu
Gly Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser 35 40 45 gtt
gat tat gat ggt gat agt tat atg aac tgg tat caa cag aaa cca 192 Val
Asp Tyr Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro 50 55
60 gga cag cca ccc aaa ctc ctc atc tat gtt gca tcc aat cta gag tct
240 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Val Ala Ser Asn Leu Glu Ser
65 70 75 80 ggg gtc cca gac agg ttt agt ggc agt ggg tct ggg aca gac
ttc acc 288 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr 85 90 95 ctc acc atc agt tct ctg cag gcg gag gat gtt gca
gtc tat tac tgt 336 Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala
Val Tyr Tyr Cys 100 105 110 cag caa agt ctt cag gac cct ccg acg ttc
ggt gga ggt acc aag gtg 384 Gln Gln Ser Leu Gln Asp Pro Pro Thr Phe
Gly Gly Gly Thr Lys Val 115 120 125 gaa atc aaa cga act gtg gct gca
cta tct gtc ttc atc ttc ccg cca 432 Glu Ile Lys Arg Thr Val Ala Ala
Leu Ser Val Phe Ile Phe Pro Pro 130 135 140 tct gat gag cag ttg aaa
tct gga act gcc tct gtt gtg tgc ctg ctg 480 Ser Asp Glu Gln Leu Lys
Ser Gly Thr Ala Ser Val Val Cys Leu Leu 145 150 155 160 aat aac ttc
tat ccc aga gag gcc aaa gta cag tgg aag gtg gat aac 528 Asn Asn Phe
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn 165 170 175 gcc
ctc caa tcg ggt aac tcc cag gag agt gtc aca gag cag gac agc 576 Ala
Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser 180 185
190 aag gac agc acc tac agc ctc agc agc acc ctg acg ctg agc aaa gca
624 Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
195 200 205 gac tac gag aaa cac aaa gtc tac gcc tgc gaa gtc acc cat
cag ggc 672 Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His
Gln Gly 210 215 220 ctg agc tcg ccc gtc aca aag agc ttc aac agg gga
gag tgt tag 717 Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys 225 230 235 11 238 PRT Artificial Sequence Description of
Artificial Sequence Synthetic TRX1 antibody light chain construct
11 Met Glu Thr Asp Thr Ile Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15 Gly Ser Thr Gly Asp Ile Val Met Thr Gln Ser Pro Asp Ser
Leu Ala 20 25 30 Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys
Ala Ser Gln Ser 35 40 45 Val Asp Tyr Asp Gly Asp Ser Tyr Met Asn
Trp Tyr Gln Gln Lys Pro 50 55 60 Gly Gln Pro Pro Lys Leu Leu Ile
Tyr Val Ala Ser Asn Leu Glu Ser 65 70 75 80 Gly Val Pro Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 85 90 95 Leu Thr Ile Ser
Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys 100 105 110 Gln Gln
Ser Leu Gln Asp Pro Pro Thr Phe Gly Gly Gly Thr Lys Val 115 120 125
Glu Ile Lys Arg Thr Val Ala Ala Leu Ser Val Phe Ile Phe Pro Pro 130
135 140 Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu
Leu 145 150 155 160 Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp
Lys Val Asp Asn 165 170 175 Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser
Val Thr Glu Gln Asp Ser 180 185 190 Lys Asp Ser Thr Tyr Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala 195 200 205 Asp Tyr Glu Lys His Lys
Val Tyr Ala Cys Glu Val Thr His Gln Gly 210 215 220 Leu Ser Ser Pro
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 12 218 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
TRX1 antibody light chain construct 12 Asp Ile Val Met Thr Gln Ser
Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile
Asn Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30 Gly Asp Ser
Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys
Leu Leu Ile Tyr Val Ala Ser Asn Leu Glu Ser Gly Val Pro Asp 50 55
60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80 Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
Ser Leu 85 90 95 Gln Asp Pro Pro Thr Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Leu Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185
190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 13 467
PRT Artificial Sequence Description of Artificial Sequence
Synthetic TRX1 antibody heavy chain construct 13 Met Glu Trp Ile
Trp Ile Phe Leu Leu Ile Leu Ser Gly Thr Arg Gly 1 5 10 15 Val Gln
Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30
Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35
40 45 Thr Ala Tyr Val Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu 50 55 60 Glu Trp Met Gly Glu Ile Tyr Pro Gly Ser Gly Ser Ser
Tyr Tyr Asn 65 70 75 80 Glu Lys Phe Lys Gly Arg Val Thr Met Thr Arg
Asp Thr Ser Thr Ser 85 90 95 Thr Val Tyr Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Ser Gly
Asp Gly Ser Arg Phe Val Tyr Trp Gly 115 120 125 Gln Gly Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 130 135 140 Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 145 150 155 160
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 165
170 175 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala 180 185 190 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val 195 200 205 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His 210 215 220 Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys 225 230 235 240 Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 245 250 255 Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290
295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr
Tyr 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro 405 410 415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460 Pro Gly Lys 465 14 1404
DNA Artificial Sequence Description of Artificial Sequence
Synthetic TRX1 antibody heavy chain construct 14 atg gaa tgg atc
tgg atc ttt ctc ctc atc ctg tca gga act cga ggt 48 Met Glu Trp Ile
Trp Ile Phe Leu Leu Ile Leu Ser Gly Thr Arg Gly 1 5 10 15 gtc cag
tcc cag gtt cag ctg gtg cag tct gga gct gaa gtg aag aag 96 Val Gln
Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30
cct ggg gct tca gtg aag gtg tcc tgt aag gct tct gga tac aca ttc 144
Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35
40 45 act gcc tat gtt ata agc tgg gtg agg cag gca cct gga cag ggc
ctt 192 Thr Ala Tyr Val Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu 50 55 60 gag tgg atg gga gag att tat cct gga agc ggt agt agt
tat tat aat 240 Glu Trp Met Gly Glu Ile Tyr Pro Gly Ser Gly Ser Ser
Tyr Tyr Asn 65 70 75 80 gag aag ttc aag ggc agg gtc aca atg act aga
gac aca tcc acc agc 288 Glu Lys Phe Lys Gly Arg Val Thr Met Thr Arg
Asp Thr Ser Thr Ser 85 90 95 aca gtc tac atg gaa ctc agc agc ctg
agg tct gag gac act gcg gtc 336 Thr Val Tyr Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val 100 105 110 tat tac tgt gca aga tcc ggg
gac ggc agt cgg ttt gtt tac tgg ggc 384 Tyr Tyr Cys Ala Arg Ser Gly
Asp Gly Ser Arg Phe Val Tyr Trp Gly 115 120 125 caa ggg aca cta gtc
aca gtc tcc tca gcc tcc acc aag ggc cca tcg 432 Gln Gly Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 130 135 140 gtc ttc ccc
ctg gca ccc tcc tcc aag agc acc tct ggg ggc aca gcg 480 Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 145 150 155 160
gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccg gtg acg gtg 528
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 165
170 175 tcg tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc ttc ccg
gct 576 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala 180 185 190 gtc cta cag tcc tca gga ctc tac tcc ctc agc agc gtg
gtg acc gtg 624 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val 195 200 205 ccc tcc agc agc ttg ggc acc cag acc tac atc
tgc aac gtg aat cac 672 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His 210 215 220 aag ccc agc aac acc aag gtg gac aag
aaa gtt gag ccc aaa tct tgt 720 Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys 225 230 235 240 gac aaa act cac aca tgc
cca ccg tgc cca gca cct gaa ctc ctg ggg 768 Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 245 250 255 gga ccg tca gtc
ttc ctc ttc ccc cca aaa ccc aag gac acc ctc atg 816 Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 atc tcc
cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc cac 864 Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285
gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg gag gtg 912
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290
295 300 cat aat gcc aag aca aag ccg cgg gag gag cag tac gcc agc acg
tac 960 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr
Tyr 305 310 315 320 cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac
tgg ctg aat ggc 1008 Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly 325 330 335 aag gag tac aag tgc aag gtc tcc aac
aaa gcc ctc cca gcc ccc atc 1056 Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile 340 345 350 gag aaa acc atc tcc aaa
gcc aaa ggg cag ccc cga gaa cca cag gtg 1104 Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 tac acc ctg
ccc cca tcc cgg gat gag ctg acc aag aac cag gtc agc 1152 Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 370 375 380
ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg gag
1200 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 385 390 395 400 tgg gag agc aat ggg cag ccg gag aac aac tac aag
acc acg cct ccc 1248 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro 405 410 415 gtg ctg gac tcc gac ggc tcc ttc ttc
ctc tac agc aag ctc acc gtg 1296 Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430 gac aag agc agg tgg cag
cag ggg aac gtc ttc tca tgc tcc gtg atg 1344 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445 cat gag gct
ctg cac aac cac tac acg cag aag agc ctc tcc ctg tct 1392 His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460
ccg ggt aaa tga 1404 Pro Gly Lys 465 15 467 PRT Artificial Sequence
Description of Artificial Sequence Synthetic TRX1 antibody heavy
chain construct 15 Met Glu Trp Ile Trp Ile Phe Leu Leu Ile Leu Ser
Gly Thr Arg Gly 1 5 10 15 Val Gln Ser Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Ala Tyr Val Ile Ser
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Met Gly
Glu Ile Tyr Pro Gly Ser Gly Ser Ser Tyr Tyr Asn 65 70 75 80 Glu Lys
Phe Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser 85 90 95
Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100
105 110 Tyr Tyr Cys Ala Arg Ser Gly Asp Gly Ser Arg Phe Val Tyr Trp
Gly 115 120 125 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser 130 135 140 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala 145 150 155 160 Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val 165 170 175 Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala 180 185 190 Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 195 200 205 Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 210 215 220
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 225
230 235 240 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly 245 250 255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His 275 280 285 Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val 290 295 300 His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr 305 310 315 320 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 325 330 335 Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 340 345
350 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
355 360 365 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445 His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460 Pro
Gly Lys 465 16 448 PRT Artificial Sequence Description of
Artificial Sequence Synthetic TRX1 antibody heavy chain construct
16 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
Ala Tyr 20 25 30 Val Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45 Gly Glu Ile Tyr Pro Gly Ser Gly Ser Ser
Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Arg
Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly
Asp Gly Ser Arg Phe Val Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130
135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250
255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr
Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375
380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys 435 440 445 17 238 PRT Artificial
Sequence Description of Artificial Sequence Synthetic TRX1 antibody
light chain sequence 17 Met Glu Thr Asp Thr Ile Leu Leu Trp Val Leu
Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ile Val Met Thr
Gln Ser Pro Asp Ser Leu Ala 20 25 30 Val Ser Leu Gly Glu Arg Ala
Thr Ile Asn Cys Lys Ala Ser Gln Ser 35 40 45 Val Asp Tyr Asp Gly
Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro 50 55 60 Gly Gln Pro
Pro Lys Leu Leu Ile Tyr Val Ala Ser Asn Leu Glu Ser 65 70 75 80 Gly
Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 85 90
95 Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys
100 105 110 Gln Gln Ser Leu Gln Asp Pro Pro Thr Phe Gly Gly Gly Thr
Lys Val 115 120 125 Glu Ile Lys Arg Thr Val Ala Ala Leu Ser Val Phe
Ile Phe Pro Pro 130 135 140 Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala
Ser Val Val Cys Leu Leu 145 150 155 160 Asn Asn Phe Tyr Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Asn 165 170 175 Ala Leu Gln Ser Gly
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser 180 185 190 Lys Asp Ser
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala 195 200 205 Asp
Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly 210 215
220 Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230
235 18 717 DNA Artificial Sequence Description of Artificial
Sequence Synthetic TRX1 antibody light chain sequence 18 atg gag
aca gac aca atc ctg cta tgg gtg ctg ctg ctc tgg gtt cca 48 Met Glu
Thr Asp Thr Ile Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15
ggc tcc act ggt gac att gtg atg acc caa tct cca gat tct ttg gct 96
Gly Ser Thr Gly Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala 20
25 30 gtg tct cta ggt gag agg gcc acc atc aac tgc aag gcc agc caa
agt 144 Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln
Ser 35 40 45 gtt gat tat gat ggt gat agt tat atg aac tgg tat caa
cag aaa cca 192 Val Asp Tyr Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln
Gln Lys Pro 50 55 60 gga cag cca ccc aaa ctc ctc atc tat gtt gca
tcc aat cta gag tct 240 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Val Ala
Ser Asn Leu Glu Ser 65 70 75 80 ggg gtc cca gac agg ttt agt ggc agt
ggg tct ggg aca gac ttc acc 288 Gly Val Pro Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr 85 90 95 ctc acc atc agt tct ctg cag
gcg gag gat gtt gca gtc tat tac tgt 336 Leu Thr Ile Ser Ser Leu Gln
Ala Glu Asp Val Ala Val Tyr Tyr Cys 100 105 110 cag caa agt ctt cag
gac cct ccg acg ttc ggt gga ggt acc aag gtg 384 Gln Gln Ser Leu Gln
Asp Pro Pro Thr Phe Gly Gly Gly Thr Lys Val 115 120 125 gaa atc aaa
cga act gtg gct gca cta tct gtc ttc atc ttc ccg cca 432 Glu Ile Lys
Arg Thr Val Ala Ala Leu Ser Val Phe Ile Phe Pro Pro 130 135 140 tct
gat gag cag ttg aaa tct gga act gcc tct gtt gtg tgc ctg ctg 480 Ser
Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu 145 150
155 160 aat aac ttc tat ccc aga gag gcc aaa gta cag tgg aag gtg gat
aac 528 Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp
Asn 165 170 175 gcc ctc caa tcg ggt aac tcc cag gag agt gtc aca gag
cag gac agc 576 Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu
Gln Asp Ser 180 185 190 aag gac agc acc tac agc ctc agc agc acc ctg
acg ctg agc aaa gca 624 Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu
Thr Leu Ser Lys Ala 195 200 205 gac tac gag aaa cac aaa gtc tac gcc
tgc gaa gtc acc cat cag ggc 672 Asp Tyr Glu Lys His Lys Val Tyr Ala
Cys Glu Val Thr His Gln Gly 210 215 220 ctg agc tcg ccc gtc aca aag
agc ttc aac agg gga gag tgt tag 717 Leu Ser Ser Pro Val Thr Lys Ser
Phe Asn Arg Gly Glu Cys 225 230 235 19 238 PRT Artificial Sequence
Description of Artificial Sequence Synthetic TRX1 antibody light
chain construct 19 Met Glu Thr Asp Thr Ile Leu Leu
Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ile
Val Met Thr Gln Ser Pro Asp Ser Leu Ala 20 25 30 Val Ser Leu Gly
Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser 35 40 45 Val Asp
Tyr Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro 50 55 60
Gly Gln Pro Pro Lys Leu Leu Ile Tyr Val Ala Ser Asn Leu Glu Ser 65
70 75 80 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr 85 90 95 Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala
Val Tyr Tyr Cys 100 105 110 Gln Gln Ser Leu Gln Asp Pro Pro Thr Phe
Gly Gly Gly Thr Lys Val 115 120 125 Glu Ile Lys Arg Thr Val Ala Ala
Leu Ser Val Phe Ile Phe Pro Pro 130 135 140 Ser Asp Glu Gln Leu Lys
Ser Gly Thr Ala Ser Val Val Cys Leu Leu 145 150 155 160 Asn Asn Phe
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn 165 170 175 Ala
Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser 180 185
190 Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
195 200 205 Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His
Gln Gly 210 215 220 Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly
Glu Cys 225 230 235 20 218 PRT Artificial Sequence Description of
Artificial Sequence Synthetic TRX1 antibody light chain construct
20 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser Val Asp
Tyr Asp 20 25 30 Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro
Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Val Ala Ser Asn Leu
Glu Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Ala Glu Asp
Val Ala Val Tyr Tyr Cys Gln Gln Ser Leu 85 90 95 Gln Asp Pro Pro
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val
Ala Ala Leu Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130
135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn
Arg Gly Glu Cys 210 215 21 467 PRT Artificial Sequence Description
of Artificial Sequence Synthetic TRX1 heavy chain construct 21 Met
Glu Trp Ile Trp Ile Phe Leu Leu Ile Leu Ser Gly Thr Arg Gly 1 5 10
15 Val Gln Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe 35 40 45 Thr Ala Tyr Val Ile Ser Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu 50 55 60 Glu Trp Met Gly Glu Ile Tyr Pro Gly Ser
Gly Ser Ser Tyr Tyr Asn 65 70 75 80 Glu Lys Phe Lys Gly Arg Val Thr
Met Thr Arg Asp Thr Ser Thr Ser 85 90 95 Thr Val Tyr Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala
Arg Ser Gly Asp Gly Ser Arg Phe Val Tyr Trp Gly 115 120 125 Gln Gly
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 130 135 140
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 145
150 155 160 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val 165 170 175 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala 180 185 190 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val 195 200 205 Pro Ser Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His 210 215 220 Lys Pro Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys 225 230 235 240 Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Ala Gly 245 250 255 Ala
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265
270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
275 280 285 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 370 375 380 Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390
395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro 405 410 415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser 450 455 460 Pro Gly Lys 465 22 1404 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
TRX1 heavy chain construct 22 atg gaa tgg atc tgg atc ttt ctc ctc
atc ctg tca gga act cga ggt 48 Met Glu Trp Ile Trp Ile Phe Leu Leu
Ile Leu Ser Gly Thr Arg Gly 1 5 10 15 gtc cag tcc cag gtt cag ctg
gtg cag tct gga gct gaa gtg aag aag 96 Val Gln Ser Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 cct ggg gct tca gtg
aag gtg tcc tgt aag gct tct gga tac aca ttc 144 Pro Gly Ala Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 act gcc tat
gtt ata agc tgg gtg agg cag gca cct gga cag ggc ctt 192 Thr Ala Tyr
Val Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 gag
tgg atg gga gag att tat cct gga agc ggt agt agt tat tat aat 240 Glu
Trp Met Gly Glu Ile Tyr Pro Gly Ser Gly Ser Ser Tyr Tyr Asn 65 70
75 80 gag aag ttc aag ggc agg gtc aca atg act aga gac aca tcc acc
agc 288 Glu Lys Phe Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr
Ser 85 90 95 aca gtc tac atg gaa ctc agc agc ctg agg tct gag gac
act gcg gtc 336 Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val 100 105 110 tat tac tgt gca aga tcc ggg gac ggc agt cgg
ttt gtt tac tgg ggc 384 Tyr Tyr Cys Ala Arg Ser Gly Asp Gly Ser Arg
Phe Val Tyr Trp Gly 115 120 125 caa ggg aca cta gtc aca gtc tcc tca
gcc tcc acc aag ggc cca tcg 432 Gln Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser 130 135 140 gtc ttc ccc ctg gca ccc tcc
tcc aag agc acc tct ggg ggc aca gcg 480 Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala 145 150 155 160 gcc ctg ggc tgc
ctg gtc aag gac tac ttc ccc gaa ccg gtg acg gtg 528 Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 165 170 175 tcg tgg
aac tca ggc gcc ctg acc agc ggc gtg cac acc ttc ccg gct 576 Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 180 185 190
gtc cta cag tcc tca gga ctc tac tcc ctc agc agc gtg gtg acc gtg 624
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 195
200 205 ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac gtg aat
cac 672 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His 210 215 220 aag ccc agc aac acc aag gtg gac aag aaa gtt gag ccc
aaa tct tgt 720 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser Cys 225 230 235 240 gac aaa act cac aca tgc cca ccg tgc cca
gca cct gaa ctc gcg ggg 768 Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Ala Gly 245 250 255 gca ccg tca gtc ttc ctc ttc ccc
cca aaa ccc aag gac acc ctc atg 816 Ala Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 atc tcc cgg acc cct gag
gtc aca tgc gtg gtg gtg gac gtg agc cac 864 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His 275 280 285 gaa gac cct gag
gtc aag ttc aac tgg tac gtg gac ggc gtg gag gtg 912 Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300 cat aat
gcc aag aca aag ccg cgg gag gag cag tac aac agc acg tac 960 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 305 310 315
320 cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg aat ggc
1008 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 325 330 335 aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca
gcc ccc atc 1056 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 340 345 350 gag aaa acc atc tcc aaa gcc aaa ggg cag
ccc cga gaa cca cag gtg 1104 Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val 355 360 365 tac acc ctg ccc cca tcc cgg
gat gag ctg acc aag aac cag gtc agc 1152 Tyr Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 370 375 380 ctg acc tgc ctg
gtc aaa ggc ttc tat ccc agc gac atc gcc gtg gag 1200 Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400
tgg gag agc aat ggg cag ccg gag aac aac tac aag acc acg cct ccc
1248 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro 405 410 415 gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag
ctc acc gtg 1296 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 420 425 430 gac aag agc agg tgg cag cag ggg aac gtc
ttc tca tgc tcc gtg atg 1344 Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 435 440 445 cat gag gct ctg cac aac cac
tac acg cag aag agc ctc tcc ctg tct 1392 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460 ccg ggt aaa tga
1404 Pro Gly Lys 465 23 467 PRT Artificial Sequence Description of
Artificial Sequence Synthetic TRX1 heavy chain construct 23 Met Glu
Trp Ile Trp Ile Phe Leu Leu Ile Leu Ser Gly Thr Arg Gly 1 5 10 15
Val Gln Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20
25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe 35 40 45 Thr Ala Tyr Val Ile Ser Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu 50 55 60 Glu Trp Met Gly Glu Ile Tyr Pro Gly Ser Gly
Ser Ser Tyr Tyr Asn 65 70 75 80 Glu Lys Phe Lys Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser 85 90 95 Thr Val Tyr Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg
Ser Gly Asp Gly Ser Arg Phe Val Tyr Trp Gly 115 120 125 Gln Gly Thr
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 130 135 140 Val
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 145 150
155 160 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val 165 170 175 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala 180 185 190 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val 195 200 205 Pro Ser Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His 210 215 220 Lys Pro Ser Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys 225 230 235 240 Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Ala Gly 245 250 255 Ala Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275
280 285 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395
400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
405 410 415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser 450 455 460 Pro Gly Lys 465 24 448 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
TRX1 heavy chain construct 24 Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Ala Tyr 20 25 30 Val Ile Ser Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile
Tyr Pro Gly Ser Gly Ser Ser Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys
Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70
75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg Ser Gly Asp Gly Ser Arg Phe Val Tyr Trp Gly
Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195
200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Ala Gly
Ala Pro Ser 225 230
235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 355
360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 25 238 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
TRX1 antibody light chain construct 25 Met Glu Thr Asp Thr Ile Leu
Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp
Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala 20 25 30 Val Ser Leu
Gly Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser 35 40 45 Val
Asp Tyr Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro 50 55
60 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Val Ala Ser Asn Leu Glu Ser
65 70 75 80 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr 85 90 95 Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala
Val Tyr Tyr Cys 100 105 110 Gln Gln Ser Leu Gln Asp Pro Pro Thr Phe
Gly Gly Gly Thr Lys Val 115 120 125 Glu Ile Lys Arg Thr Val Ala Ala
Pro Ser Val Phe Ile Phe Pro Pro 130 135 140 Ser Asp Glu Gln Leu Lys
Ser Gly Thr Ala Ser Val Val Cys Leu Leu 145 150 155 160 Asn Asn Phe
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn 165 170 175 Ala
Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser 180 185
190 Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
195 200 205 Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His
Gln Gly 210 215 220 Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly
Glu Cys 225 230 235 26 717 DNA Artificial Sequence Description of
Artificial Sequence Synthetic TRX1 antibody light chain construct
26 atg gag aca gac aca atc ctg cta tgg gtg ctg ctg ctc tgg gtt cca
48 Met Glu Thr Asp Thr Ile Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15 ggc tcc act ggt gac att gtg atg acc caa tct cca gat tct
ttg gct 96 Gly Ser Thr Gly Asp Ile Val Met Thr Gln Ser Pro Asp Ser
Leu Ala 20 25 30 gtg tct cta ggt gag agg gcc acc atc aac tgc aag
gcc agc caa agt 144 Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys
Ala Ser Gln Ser 35 40 45 gtt gat tat gat ggt gat agt tat atg aac
tgg tat caa cag aaa cca 192 Val Asp Tyr Asp Gly Asp Ser Tyr Met Asn
Trp Tyr Gln Gln Lys Pro 50 55 60 gga cag cca ccc aaa ctc ctc atc
tat gtt gca tcc aat cta gag tct 240 Gly Gln Pro Pro Lys Leu Leu Ile
Tyr Val Ala Ser Asn Leu Glu Ser 65 70 75 80 ggg gtc cca gac agg ttt
agt ggc agt ggg tct ggg aca gac ttc acc 288 Gly Val Pro Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 85 90 95 ctc acc atc agt
tct ctg cag gcg gag gat gtt gca gtc tat tac tgt 336 Leu Thr Ile Ser
Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys 100 105 110 cag caa
agt ctt cag gac cct ccg acg ttc ggt gga ggt acc aag gtg 384 Gln Gln
Ser Leu Gln Asp Pro Pro Thr Phe Gly Gly Gly Thr Lys Val 115 120 125
gaa atc aaa cga act gtg gct gca cca tct gtc ttc atc ttc ccg cca 432
Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro 130
135 140 tct gat gag cag ttg aaa tct gga act gcc tct gtt gtg tgc ctg
ctg 480 Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu
Leu 145 150 155 160 aat aac ttc tat ccc aga gag gcc aaa gta cag tgg
aag gtg gat aac 528 Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp
Lys Val Asp Asn 165 170 175 gcc ctc caa tcg ggt aac tcc cag gag agt
gtc aca gag cag gac agc 576 Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser
Val Thr Glu Gln Asp Ser 180 185 190 aag gac agc acc tac agc ctc agc
agc acc ctg acg ctg agc aaa gca 624 Lys Asp Ser Thr Tyr Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala 195 200 205 gac tac gag aaa cac aaa
gtc tac gcc tgc gaa gtc acc cat cag ggc 672 Asp Tyr Glu Lys His Lys
Val Tyr Ala Cys Glu Val Thr His Gln Gly 210 215 220 ctg agc tcg ccc
gtc aca aag agc ttc aac agg gga gag tgt tag 717 Leu Ser Ser Pro Val
Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 27 238 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
TRX1 light chain construct 27 Met Glu Thr Asp Thr Ile Leu Leu Trp
Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ile Val
Met Thr Gln Ser Pro Asp Ser Leu Ala 20 25 30 Val Ser Leu Gly Glu
Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser 35 40 45 Val Asp Tyr
Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro 50 55 60 Gly
Gln Pro Pro Lys Leu Leu Ile Tyr Val Ala Ser Asn Leu Glu Ser 65 70
75 80 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr 85 90 95 Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val
Tyr Tyr Cys 100 105 110 Gln Gln Ser Leu Gln Asp Pro Pro Thr Phe Gly
Gly Gly Thr Lys Val 115 120 125 Glu Ile Lys Arg Thr Val Ala Ala Pro
Ser Val Phe Ile Phe Pro Pro 130 135 140 Ser Asp Glu Gln Leu Lys Ser
Gly Thr Ala Ser Val Val Cys Leu Leu 145 150 155 160 Asn Asn Phe Tyr
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn 165 170 175 Ala Leu
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser 180 185 190
Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala 195
200 205 Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln
Gly 210 215 220 Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys 225 230 235 28 218 PRT Artificial Sequence Description of
Artificial Sequence Synthetic TRX1 light chain construct 28 Asp Ile
Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp 20
25 30 Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro
Pro 35 40 45 Lys Leu Leu Ile Tyr Val Ala Ser Asn Leu Glu Ser Gly
Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Ala Glu Asp Val Ala Val
Tyr Tyr Cys Gln Gln Ser Leu 85 90 95 Gln Asp Pro Pro Thr Phe Gly
Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150
155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys 210 215 29 467 PRT Artificial Sequence Description of
Artificial Sequence Synthetic TRX1 heavy chain construct 29 Met Glu
Trp Ile Trp Ile Phe Leu Leu Ile Leu Ser Gly Thr Arg Gly 1 5 10 15
Val Gln Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20
25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe 35 40 45 Thr Ala Tyr Val Ile Ser Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu 50 55 60 Glu Trp Met Gly Glu Ile Tyr Pro Gly Ser Gly
Ser Ser Tyr Tyr Asn 65 70 75 80 Glu Lys Phe Lys Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser 85 90 95 Thr Val Tyr Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg
Ser Gly Asp Gly Ser Arg Phe Val Tyr Trp Gly 115 120 125 Gln Gly Thr
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 130 135 140 Val
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 145 150
155 160 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val 165 170 175 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala 180 185 190 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val 195 200 205 Pro Ser Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His 210 215 220 Lys Pro Ser Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys 225 230 235 240 Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 245 250 255 Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275
280 285 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala
Ser Thr Tyr 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395
400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
405 410 415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser 450 455 460 Pro Gly Lys 465 30 1404 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
TRX1 heavy chain construct 30 atg gaa tgg atc tgg atc ttt ctc ctc
atc ctg tca gga act cga ggt 48 Met Glu Trp Ile Trp Ile Phe Leu Leu
Ile Leu Ser Gly Thr Arg Gly 1 5 10 15 gtc cag tcc cag gtt cag ctg
gtg cag tct gga gct gaa gtg aag aag 96 Val Gln Ser Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 cct ggg gct tca gtg
aag gtg tcc tgt aag gct tct gga tac aca ttc 144 Pro Gly Ala Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 act gcc tat
gtt ata agc tgg gtg agg cag gca cct gga cag ggc ctt 192 Thr Ala Tyr
Val Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 gag
tgg atg gga gag att tat cct gga agc ggt agt agt tat tat aat 240 Glu
Trp Met Gly Glu Ile Tyr Pro Gly Ser Gly Ser Ser Tyr Tyr Asn 65 70
75 80 gag aag ttc aag ggc agg gtc aca atg act aga gac aca tcc acc
agc 288 Glu Lys Phe Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr
Ser 85 90 95 aca gtc tac atg gaa ctc agc agc ctg agg tct gag gac
act gcg gtc 336 Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val 100 105 110 tat tac tgt gca aga tcc ggg gac ggc agt cgg
ttt gtt tac tgg ggc 384 Tyr Tyr Cys Ala Arg Ser Gly Asp Gly Ser Arg
Phe Val Tyr Trp Gly 115 120 125 caa ggg aca cta gtc aca gtc tcc tca
gcc tcc acc aag ggc cca tcg 432 Gln Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser 130 135 140 gtc ttc ccc ctg gca ccc tcc
tcc aag agc acc tct ggg ggc aca gcg 480 Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala 145 150 155 160 gcc ctg ggc tgc
ctg gtc aag gac tac ttc ccc gaa ccg gtg acg gtg 528 Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 165 170 175 tcg tgg
aac tca ggc gcc ctg acc agc ggc gtg cac acc ttc ccg gct 576 Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 180 185 190
gtc cta cag tcc tca gga ctc tac tcc ctc agc agc gtg gtg acc gtg 624
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 195
200 205 ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac gtg aat
cac 672 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His 210 215 220 aag ccc agc aac acc aag gtg gac aag aaa gtt gag ccc
aaa tct tgt 720 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser Cys 225 230 235 240 gac aaa act cac aca tgc cca ccg tgc cca
gca cct gaa ctc ctg ggg 768 Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly 245 250 255 gga ccg tca gtc ttc ctc ttc ccc
cca aaa ccc aag gac acc ctc atg 816 Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 atc tcc cgg acc cct gag
gtc aca tgc gtg gtg gtg gac gtg agc cac 864 Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His 275 280 285 gaa gac cct gag
gtc aag ttc aac tgg tac gtg gac ggc gtg gag gtg 912 Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300 cat aat
gcc aag aca aag ccg cgg gag gag cag tac gcc agc acg tac 960 His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr 305 310 315
320 cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg aat ggc
1008 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly 325 330 335 aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca
gcc ccc atc 1056 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 340 345 350 gag aaa acc atc tcc aaa gcc aaa ggg cag
ccc cga gaa cca cag gtg 1104 Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val 355 360 365 tac acc ctg ccc cca tcc cgg
gat gag ctg acc aag aac cag gtc agc 1152 Tyr Thr Leu Pro Pro Ser
Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser 370 375 380 ctg acc tgc ctg gtc
aaa ggc ttc tat ccc agc gac atc gcc gtg gag 1200 Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 tgg
gag agc aat ggg cag ccg gag aac aac tac aag acc acg cct ccc 1248
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405
410 415 gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc
gtg 1296 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val 420 425 430 gac aag agc agg tgg cag cag ggg aac gtc ttc tca
tgc tcc gtg atg 1344 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met 435 440 445 cat gag gct ctg cac aac cac tac acg
cag aag agc ctc tcc ctg tct 1392 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460 ccg ggt aaa tga 1404
Pro Gly Lys 465 31 467 PRT Artificial Sequence Description of
Artificial Sequence Synthetic TRX1 heavy chain construct 31 Met Glu
Trp Ile Trp Ile Phe Leu Leu Ile Leu Ser Gly Thr Arg Gly 1 5 10 15
Val Gln Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20
25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe 35 40 45 Thr Ala Tyr Val Ile Ser Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu 50 55 60 Glu Trp Met Gly Glu Ile Tyr Pro Gly Ser Gly
Ser Ser Tyr Tyr Asn 65 70 75 80 Glu Lys Phe Lys Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser 85 90 95 Thr Val Tyr Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg
Ser Gly Asp Gly Ser Arg Phe Val Tyr Trp Gly 115 120 125 Gln Gly Thr
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 130 135 140 Val
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 145 150
155 160 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val 165 170 175 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala 180 185 190 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val 195 200 205 Pro Ser Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His 210 215 220 Lys Pro Ser Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys 225 230 235 240 Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 245 250 255 Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275
280 285 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala
Ser Thr Tyr 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395
400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
405 410 415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser 450 455 460 Pro Gly Lys 465 32 448 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
TRX1 heavy chain construct 32 Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Ala Tyr 20 25 30 Val Ile Ser Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile
Tyr Pro Gly Ser Gly Ser Ser Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys
Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70
75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg Ser Gly Asp Gly Ser Arg Phe Val Tyr Trp Gly
Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195
200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val 290 295 300 Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315
320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu 340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
445 33 451 PRT Artificial Sequence Description of Artificial
Sequence Humanized CD8 antibody heavy chain construct 33 Gln Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Phe 20
25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ala Leu Ile Tyr Tyr Asp Gly Ser Asn Lys Phe Tyr Ala
Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Pro His Tyr Asp Gly
Tyr Tyr His Phe Phe Asp Ser Trp Gly 100 105 110 Gln Gly Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150
155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275
280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395
400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 34 213 PRT Artificial
Sequence Description of Artificial Sequence Humanized CD8 antibody
light chain construct 34 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys
Gly Ser Gln Asp Ile Asn Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asn Thr Asp
Ile Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Tyr Gln Tyr Asn Asn Gly Tyr Thr 85
90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly Asn Ser Gln Glu 145 150 155 160 Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205
Asn Arg Gly Glu Cys 210 35 135 PRT Homo sapiens 35 Met Val Leu Gln
Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala
Tyr Gly Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala 20 25 30
Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser 35
40 45 Leu Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln
Gln 50 55 60 Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala
Ser Thr Arg 65 70 75 80 Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp 85 90 95 Phe Thr Leu Thr Ile Ser Ser Leu Gln
Ala Glu Asp Val Ala Val Tyr 100 105 110 Tyr Cys Gln Gln Tyr Tyr Ser
Thr Pro Pro Met Phe Gly Gln Gly Thr 115 120 125 Lys Val Glu Ile Lys
Arg Thr 130 135 36 142 PRT Homo sapiens 36 Leu Leu Ala Val Ala Pro
Gly Ala His Ser Gln Val Gln Leu Val Gln 1 5 10 15 Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys 20 25 30 Lys Ala
Ser Gly Tyr Thr Phe Thr Asn Tyr Tyr Met His Trp Val Arg 35 40 45
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Ser 50
55 60 Gly Asn Ser Thr Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr
Met 65 70 75 80 Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu Leu
Ser Ser Leu 85 90 95 Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala
Arg Glu Lys Leu Ala 100 105 110 Thr Thr Ile Phe Gly Val Leu Ile Ile
Thr Gly Met Asp Tyr Trp Gly 115 120 125 Gln Gly Thr Leu Val Thr Val
Ser Ser Gly Ser Ala Ser Ala 130 135 140 37 76 DNA Artificial
Sequence Description of Artificial Sequence Synthetic primer 37
tgacattgtg atgacccaat ctccagattc tttggctgtg tctctaggtg agagggccac
60 catcaactgc aaggcc 76 38 29 DNA Artificial Sequence Description
of Artificial Sequence Synthetic primer 38 tgaactggta tcaacagaaa
ccaggacag 29 39 28 DNA Artificial Sequence Description of
Artificial Sequence Synthetic primer 39 agagtctggg gtcccagaca
ggtttagt 28 40 42 DNA Artificial Sequence Description of Artificial
Sequence Synthetic primer 40 gtcttcagga ccctccgacg ttcggtggag
gtaccaagct gg 42 41 52 DNA Artificial Sequence Description of
Artificial Sequence Synthetic primer 41 caccctcacc atcagttctc
tgcaggcgga ggatgttgca gtctattagt gt 52 42 24 DNA Artificial
Sequence Description of Artificial Sequence Synthetic primer 42
agctttacag ttactgagca caca 24 43 24 DNA Artificial Sequence
Description of Artificial Sequence Synthetic primer 43 tcgatgtgtg
ctcagtaact gtaa 24 44 75 DNA Artificial Sequence Description of
Artificial Sequence Synthetic primer 44 ggttcagctg gtgcagtctg
gagctgaagt gaagaagcct ggggcttcag tgaaggtgtc 60 ctgtaaggct tctgg 75
45 52 DNA Artificial Sequence Description of Artificial Sequence
Synthetic primer 45 agctgggtga ggcaggcacc tggacagggc cttgagtgga
tgggagagat tt 52 46 60 DNA Artificial Sequence Description of
Artificial Sequence Synthetic primer 46 caagggcagg gtcacaatga
ctagagacac atccaccagc acagtctaca tggaactcag 60 47 43 DNA Artificial
Sequence Description of Artificial Sequence Synthetic primer 47
cagcctgagg tctgaggaca ctgcggtcta ttactgtgca aga 43 48 24 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
primer 48 gccaagggac actagtcact gtgt 24 49 39 DNA Artificial
Sequence Description of Artificial Sequence Synthetic primer 49
actctaacca tggaatggat ctggatcttt ctcctcatc 39 50 39 DNA Artificial
Sequence Description of Artificial Sequence Synthetic primer 50
tcactgccta tgttataagc tgggtgaggc aggcacctg 39 51 21 DNA Artificial
Sequence Description of Artificial Sequence Synthetic primer 51
actagtcaca gtctcctcag c 21 52 22 DNA Artificial Sequence
Description of Artificial Sequence Synthetic primer 52
gaattcattt
acccggagac ag 22 53 49 DNA Artificial Sequence Description of
Artificial Sequence Synthetic primer 53 ccgtgcccag cacctgaact
cgcgggggca ccgtcagtct tcctccccc 49 54 26 DNA Artificial Sequence
Description of Artificial Sequence Synthetic primer 54 ggtaccaagg
tggaaatcaa acgaac 26 55 25 DNA Artificial Sequence Description of
Artificial Sequence Synthetic primer 55 aagcttctaa cactctcccc tgttg
25 56 78 DNA Artificial Sequence Description of Artificial Sequence
Synthetic primer 56 aagcttatgg aatggatctg gatctttctc ctcatcctgt
caggaactcg aggtgtccag 60 tcccaggttc agctggtg 78 57 74 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
primer 57 ctgtaaggct tctggataca cattcactgc ctatgttata agctgggtga
ggcaggcacc 60 tggacagggc cttg 74 58 70 DNA Artificial Sequence
Description of Artificial Sequence Synthetic primer 58 ggtagtagtt
attataatga gaagttcaag ggcagggtca caatgactag agacacatcc 60
accagcacag 70 59 77 DNA Artificial Sequence Description of
Artificial Sequence Synthetic primer 59 gaggacactg cggtctatta
ctgtgcaaga tccggggacg gcagtcggtt tgtttactgg 60 ggccaaggga cactagt
77 60 90 DNA Artificial Sequence Description of Artificial Sequence
Synthetic primer 60 gtgtatccag aagccttaca ggacaccttc actgaagccc
caggcttctt cacttcagct 60 ccagactgca ccagctgaac ctgggactgg 90 61 77
DNA Artificial Sequence Description of Artificial Sequence
Synthetic primer 61 cttctcatta taataactac taccgcttcc aggataaatc
tctcccatcc actcaaggcc 60 ctgtccaggt gcctgcc 77 62 71 DNA Artificial
Sequence Description of Artificial Sequence Synthetic primer 62
gtaatagacc gcagtgtcct cagacctcag gctgctgagt tccatgtaga ctgtgctggt
60 ggatgtgtct c 71 63 69 DNA Artificial Sequence Description of
Artificial Sequence Synthetic primer 63 gaattcatgg agacagacac
aatcctgcta tgggtgctgc tgctctgggt tccaggctcc 60 actggtgac 69 64 66
DNA Artificial Sequence Description of Artificial Sequence
Synthetic primer 64 ggctgtgtct ctaggtgaga gggccaccat caactgcaag
gccagccaaa gtgttgatta 60 tgatgg 66 65 65 DNA Artificial Sequence
Description of Artificial Sequence Synthetic primer 65 cagaaaccag
gacagccacc caaactcctc atctatgttg catccaatct agagtctggg 60 gtccc 65
66 66 DNA Artificial Sequence Description of Artificial Sequence
Synthetic primer 66 ggacagactt caccctcacc atcagttctc tgcaggcgga
ggatgttgca gtctattact 60 gtcagc 66 67 67 DNA Artificial Sequence
Description of Artificial Sequence Synthetic primer 67 cacctagaga
cacagccaaa gaatctggag attgggtcat cacaatgtca ccagtggagc 60 ctggaac
67 68 66 DNA Artificial Sequence Description of Artificial Sequence
Synthetic primer 68 ggtggctgtc ctggtttctg ttgataccag ttcatataac
tatcaccatc ataatcaaca 60 ctttgg 66 69 67 DNA Artificial Sequence
Description of Artificial Sequence Synthetic primer 69 ggtgagggtg
aagtctgtcc cagacccact gccactaaac ctgtctggga ccccagactc 60 tagattg
67 70 60 DNA Artificial Sequence Description of Artificial Sequence
Synthetic primer 70 ggtacctcca ccgaacgtcg gagggtcctg aagactttgc
tgacagtaat agactgcaac 60 71 1404 DNA Artificial Sequence
Description of Artificial Sequence Synthetic TRX1 antibody heavy
chain construct 71 atg gaa tgg atc tgg atc ttt ctc ctc atc ctg tca
gga act gca ggt 48 Met Glu Trp Ile Trp Ile Phe Leu Leu Ile Leu Ser
Gly Thr Ala Gly 1 5 10 15 gtc cag tcc cag gtt cag ctg gtg cag tct
gga gct gaa gtg aag aag 96 Val Gln Ser Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys 20 25 30 cct ggg gct tca gtg aag gtg tcc
tgt aag gct tct gga tac aca ttc 144 Pro Gly Ala Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 act gcc tat gtt ata agc
tgg gtg agg cag gca cct gga cag ggc ctt 192 Thr Ala Tyr Val Ile Ser
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 gag tgg atg gga
gag att tat cct gga agc ggt agt agt tat tat aat 240 Glu Trp Met Gly
Glu Ile Tyr Pro Gly Ser Gly Ser Ser Tyr Tyr Asn 65 70 75 80 gag aag
ttc aag ggc agg gtc aca atg act aga gac aca tcc acc agc 288 Glu Lys
Phe Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser 85 90 95
aca gtc tac atg gaa ctc agc agc ctg agg tct gag gac act gcg gtc 336
Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100
105 110 tat tac tgt gca aga tcc ggg gac ggc agt cgg ttt gtt tac tgg
ggc 384 Tyr Tyr Cys Ala Arg Ser Gly Asp Gly Ser Arg Phe Val Tyr Trp
Gly 115 120 125 caa ggg aca cta gtc aca gtc tcc tca gcc tcc acc aag
ggc cca tcg 432 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser 130 135 140 gtc ttc ccc ctg gca ccc tcc tcc aag agc acc
tct ggg ggc aca gcg 480 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala 145 150 155 160 gcc ctg ggc tgc ctg gtc aag gac
tac ttc ccc gaa ccg gtg acg gtg 528 Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val 165 170 175 tcg tgg aac tca ggc gcc
ctg acc agc ggc gtg cac acc ttc ccg gct 576 Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala 180 185 190 gtc cta cag tcc
tca gga ctc tac tcc ctc agc agc gtg gtg acc gtg 624 Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 195 200 205 ccc tcc
agc agc ttg ggc acc cag acc tac atc tgc aac gtg aat cac 672 Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 210 215 220
aag ccc agc aac acc aag gtg gac aag aaa gtt gag ccc aaa tct tgt 720
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 225
230 235 240 gac aaa act cac aca tgc cca ccg tgc cca gca cct gaa ctc
ctg ggg 768 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly 245 250 255 gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag
gac acc ctc atg 816 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met 260 265 270 atc tcc cgg acc cct gag gtc aca tgc gtg
gtg gtg gac gtg agc cac 864 Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His 275 280 285 gaa gac cct gag gtc aag ttc aac
tgg tac gtg gac ggc gtg gag gtg 912 Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val 290 295 300 cat aat gcc aag aca aag
ccg cgg gag gag cag tac gcc agc acg tac 960 His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr 305 310 315 320 cgt gtg gtc
agc gtc ctc acc gtc ctg cac cag gac tgg ctg aat ggc 1008 Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 325 330 335
aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc atc
1056 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile 340 345 350 gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa
cca cag gtg 1104 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val 355 360 365 tac acc ctg ccc cca tcc cgg gat gag ctg
acc aag aac cag gtc agc 1152 Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser 370 375 380 ctg acc tgc ctg gtc aaa ggc
ttc tat ccc agc gac atc gcc gtg gag 1200 Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 tgg gag agc
aat ggg cag ccg gag aac aac tac aag acc acg cct ccc 1248 Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415
gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc gtg
1296 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val 420 425 430 gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc
tcc gtg atg 1344 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met 435 440 445 cat gag gct ctg cac aac cac tac acg cag
aag agc ctc tcc ctg tct 1392 His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser 450 455 460 ccg ggt aaa tga 1404 Pro
Gly Lys 465 72 467 PRT Artificial Sequence Description of
Artificial Sequence Synthetic TRX1 antibody heavy chain construct
72 Met Glu Trp Ile Trp Ile Phe Leu Leu Ile Leu Ser Gly Thr Ala Gly
1 5 10 15 Val Gln Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe 35 40 45 Thr Ala Tyr Val Ile Ser Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Met Gly Glu Ile Tyr Pro
Gly Ser Gly Ser Ser Tyr Tyr Asn 65 70 75 80 Glu Lys Phe Lys Gly Arg
Val Thr Met Thr Arg Asp Thr Ser Thr Ser 85 90 95 Thr Val Tyr Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr
Cys Ala Arg Ser Gly Asp Gly Ser Arg Phe Val Tyr Trp Gly 115 120 125
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 130
135 140 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala 145 150 155 160 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val 165 170 175 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala 180 185 190 Val Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val 195 200 205 Pro Ser Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His 210 215 220 Lys Pro Ser Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 225 230 235 240 Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 245 250
255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
260 265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 275 280 285 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Ala Ser Thr Tyr 305 310 315 320 Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 370 375
380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro 405 410 415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460 Pro Gly Lys 465 73
717 DNA Artificial Sequence Description of Artificial Sequence
Synthetic TRX1 antibody light chain construct 73 atg gag aca gac
aca atc ctg cta tgg gtg ctg ctg ctc tgg gtt cca 48 Met Glu Thr Asp
Thr Ile Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 ggc tcc
act ggt gac att gtg atg acc caa tct cca gat tct ttg gct 96 Gly Ser
Thr Gly Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala 20 25 30
gtg tct cta ggt gag agg gcc acc atc aac tgc aag gcc agc caa agt 144
Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser 35
40 45 gtt gat tat gat ggt gat agt tat atg aac tgg tat caa cag aaa
cca 192 Val Asp Tyr Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys
Pro 50 55 60 gga cag cca ccc aaa ctc ctc atc tat gtt gca tcc aat
cta gag tct 240 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Val Ala Ser Asn
Leu Glu Ser 65 70 75 80 ggg gtc cca gac agg ttt agt ggc agt ggg tct
ggg aca gac ttc acc 288 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr 85 90 95 ctc acc atc agt tct ctg cag gcg gag
gat gtt gca gtc tat tac tgt 336 Leu Thr Ile Ser Ser Leu Gln Ala Glu
Asp Val Ala Val Tyr Tyr Cys 100 105 110 cag caa agt ctt cag gac cct
ccg acg ttc ggt gga ggt acc aag gtg 384 Gln Gln Ser Leu Gln Asp Pro
Pro Thr Phe Gly Gly Gly Thr Lys Val 115 120 125 gaa atc aaa cga act
gtg gct gca cta tct gtc ttc atc ttc ccg cca 432 Glu Ile Lys Arg Thr
Val Ala Ala Leu Ser Val Phe Ile Phe Pro Pro 130 135 140 tct gat gag
cag ttg aaa tct gga act gcc tct gtt gtg tgc ctg ctg 480 Ser Asp Glu
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu 145 150 155 160
aat aac ttc tat ccc aga gag gcc aaa gta cag tgg aag gtg gat aac 528
Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn 165
170 175 gcc ctc caa tcg ggt aac tcc cag gag agt gtc aca gag cag gac
agc 576 Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser 180 185 190 aag gac agc acc tac agc ctc agc agc acc ctg acg ctg
agc aaa gca 624 Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala 195 200 205 gac tac gag aaa cac aaa gtc tac gcc tgc gaa
gtc acc cat cag ggc 672 Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly 210 215 220 ctg agc tcg ccc gtc aca aag agc ttc
aac agg gga gag tgt tag 717 Leu Ser Ser Pro Val Thr Lys Ser Phe Asn
Arg Gly Glu Cys 225 230 235 74 238 PRT Artificial Sequence
Description of Artificial Sequence Synthetic TRX1 antibody light
chain construct 74 Met Glu Thr Asp Thr Ile Leu Leu Trp Val Leu Leu
Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ile Val Met Thr Gln
Ser Pro Asp Ser Leu Ala 20 25 30 Val Ser Leu Gly Glu Arg Ala Thr
Ile Asn Cys Lys Ala Ser Gln Ser 35 40 45 Val Asp Tyr Asp Gly Asp
Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro 50 55 60 Gly Gln Pro Pro
Lys Leu Leu Ile Tyr Val Ala Ser Asn Leu Glu Ser 65 70 75 80 Gly Val
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 85 90 95
Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys 100
105 110 Gln Gln Ser Leu Gln Asp Pro Pro Thr Phe Gly Gly Gly Thr Lys
Val 115 120 125 Glu Ile Lys Arg Thr Val Ala Ala Leu Ser Val Phe Ile
Phe Pro Pro 130 135 140 Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser
Val Val Cys Leu Leu 145 150 155 160 Asn Asn Phe Tyr Pro Arg Glu Ala
Lys Val Gln Trp Lys Val Asp Asn 165 170 175 Ala Leu Gln Ser Gly Asn
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser 180 185 190 Lys Asp Ser Thr
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala 195 200 205 Asp Tyr
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly 210 215 220
Leu Ser Ser Pro Val
Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 75 1356 DNA
Artificial Sequence Description of Artificial Sequence Humanized
CD8 antibody heavy chain construct 75 cag gtt caa ttg gtg gag tct
gga gga ggc gtt gta cag cct gga agg 48 Gln Val Gln Leu Val Glu Ser
Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 tcc ctg aga ctc tca
tgt gca gct tct gga ttc act ttc agt gac ttt 96 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Phe 20 25 30 ggc atg aac
tgg gtt cga cag gct ccc ggg aag ggg ctg gaa tgg gtg 144 Gly Met Asn
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 gca
ctg att tac tat gat ggt agt aac aag ttc tat gca gac tct gtg 192 Ala
Leu Ile Tyr Tyr Asp Gly Ser Asn Lys Phe Tyr Ala Asp Ser Val 50 55
60 aag ggt cga ttc acc atc tcc agg gac aat tct aag aac acc cta tac
240 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 ctg caa atg aac agc ctg aga gct gag gac aca gcc gtg tat
tac tgt 288 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 gca aaa ccc cac tat gat ggt tat tat cac ttc ttt
gat tcc tgg ggc 336 Ala Lys Pro His Tyr Asp Gly Tyr Tyr His Phe Phe
Asp Ser Trp Gly 100 105 110 caa ggg aca cta gtc aca gtc tcc tca gcc
tcc acc aag ggc cca tcg 384 Gln Gly Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser 115 120 125 gtc ttc ccc ctg gca ccc tcc tcc
aag agc acc tct ggg ggc aca gcg 432 Val Phe Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 gcc ctg ggc tgc ctg gtc
aag gac tac ttc ccc gaa ccg gtg acg gtg 480 Ala Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 tcg tgg aac
tca ggc gcc ctg acc agc ggc gtg cac acc ttc ccg gct 528 Ser Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 gtc
cta cag tcc tca gga ctc tac tcc ctc agc agc gtg gtg acc gtg 576 Val
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185
190 ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac gtg aat cac
624 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
195 200 205 aag ccc agc aac acc aag gtg gac aag aaa gtt gag ccc aaa
tct tgt 672 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys 210 215 220 gac aaa act cac aca tgc cca ccg tgc cca gca cct
gaa ctc ctg ggg 720 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly 225 230 235 240 gga ccg tca gtc ttc ctc ttc ccc cca
aaa ccc aag gac acc ctc atg 768 Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met 245 250 255 atc tcc cgg acc cct gag gtc
aca tgc gtg gtg gtg gac gtg agc cac 816 Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His 260 265 270 gaa gac cct gag gtc
aag ttc aac tgg tac gtg gac ggc gtg gag gtg 864 Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 cat aat gcc
aag aca aag ccg cgg gag gag cag tac aac agc acg tac 912 His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 cgt
gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg aat ggc 960 Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310
315 320 aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc
atc 1008 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile 325 330 335 gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga
gaa cca cag gtg 1056 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 340 345 350 tac acc ctg ccc cca tcc cgg gat gag
ctg acc aag aac cag gtc agc 1104 Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 ctg acc tgc ctg gtc aaa
ggc ttc tat ccc agc gac atc gcc gtg gag 1152 Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 tgg gag agc
aat ggg cag ccg gag aac aac tac aag acc acg cct ccc 1200 Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395
400 gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc gtg
1248 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val 405 410 415 gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc
tcc gtg atg 1296 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met 420 425 430 cat gag gct ctg cac aac cac tac acg cag
aag agc ctc tcc ctg tct 1344 His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser 435 440 445 ccg ggt aaa tga 1356 Pro
Gly Lys 450 76 642 DNA Artificial Sequence Description of
Artificial Sequence Humanized CD8 antibody light chain construct 76
gac atc cag atg acc cag agc cca agc agc ctg agc gcc agc gtg ggt 48
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5
10 15 gac aga gtg acc atc acc tgt aaa gga agt cag gat att aac aat
tac 96 Asp Arg Val Thr Ile Thr Cys Lys Gly Ser Gln Asp Ile Asn Asn
Tyr 20 25 30 tta gcc tgg tac cag cag aag cca ggt aag gct cca aag
ctg ctg atc 144 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 tac aat aca gac att ttg cac acg ggt gtg cca
agc aga ttc agc ggt 192 Tyr Asn Thr Asp Ile Leu His Thr Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 agc ggt agc ggt acc gac ttc acc ttc
acc atc agc agc ctc cag cca 240 Ser Gly Ser Gly Thr Asp Phe Thr Phe
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 gag gac atc gcc acc tac tac
tgc tat cag tat aac aac ggg tac acg 288 Glu Asp Ile Ala Thr Tyr Tyr
Cys Tyr Gln Tyr Asn Asn Gly Tyr Thr 85 90 95 ttc ggc caa ggg acc
aag gtg gaa atc aaa cga act gtg gct gca cca 336 Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 tct gtc ttc
atc ttc ccg cca tct gat gag cag ttg aaa tct gga act 384 Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 gcc
tct gtt gtg tgc ctg ctg aat aac ttc tat ccc aga gag gcc aaa 432 Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135
140 gta cag tgg aag gtg gat aac gcc ctc caa tcg ggt aac tcc cag gag
480 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
145 150 155 160 agt gtc aca gag cag gac agc aag gac agc acc tac agc
ctc agc agc 528 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
Leu Ser Ser 165 170 175 acc ctg acg ctg agc aaa gca gac tac gag aaa
cac aaa gtc tac gcc 576 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
His Lys Val Tyr Ala 180 185 190 tgc gaa gtc acc cat cag ggc ctg agc
tcg ccc gtc aca aag agc ttc 624 Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro Val Thr Lys Ser Phe 195 200 205 aac agg gga gag tgt tag 642
Asn Arg Gly Glu Cys 210
* * * * *